AAV capsids for targeting human transferrin receptor

Abstract

The present disclosure pertains to targeting moieties, (e.g., CNS-targeting moieties) comprising peptides which can be inserted in the capsid of a recombinant adeno-associated virus (rAAV) vector. Also disclosed herein are compositions comprising CNS-targeting moieties disclosed herein and methods of making and using the same.

Description

Attorney Docket No.: 2011256-2513 (P1829PCT01) AAV CAPSIDS FOR TARGETING HUMAN TRANSFERRIN RECEPTOR CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 570,560, filed on March 27, 2024, the entire contents of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said sequence listing, created on March 24, 2025 is named 2011256-2513.xml and is 1,348,057 bytes in size. BACKGROUND

[0003] Targeted delivery of payloads (e.g., using recombinant adeno-associated viruses) to cells or tissues for treating and / or preventing diseases remains a challenge. SUMMARY

[0004] The present disclosure identifies certain challenges with existing targeted delivery of payloads. For example, the present disclosure identifies that a lack of targeting moieties that can specifically deliver payloads to a target cell or tissue with decreased non-specific delivery to other cells or tissues is a key challenge. Another challenge identified by the present disclosure is the poor potency associated with delivery vectors, e.g., recombinant adeno-associated virus vectors. In some embodiments, improving the potency of delivery vectors could be beneficial in obtaining clinically relevant outcomes.

[0005] Among other things, the present disclosure provides technologies that can address certain limitations identified in existing targeted delivery of payloads. The technologies provided herein are particularly useful for specifically delivering payloads to a target cell or tissue. In some embodiments, by specifically delivering payloads to a target cell or tissue, technologies provided herein can also increase the potency of a payload. In some embodiments, a target cell or tissue expresses one or more receptors (e.g., a human transferrin receptor (hTfR1)) recognized by Page 1 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) a variant AAV capsid protein disclosed herein. In some embodiments, a target cell or tissue is a central nervous system (CNS) cell and / or tissue.

[0006] The present disclosure encompasses peptides comprising targeting moieties which recognize (e.g., bind to) a hTfR1 or a variant or a fragment thereof. Also disclosed herein are recombinant adeno-associated virus (rAAV) particles comprising a variant AAV capsid protein comprising a targeting moiety which recognizes (e.g., binds to) a hTfR1 or a variant or a fragment thereof. In some embodiments, a targeting moiety in a variant AAV capsid protein is also referred to as a “peptide insertion.” In some embodiments, a targeting moiety binds to and / or recognizes a hTfR1 or a variant or a fragment thereof expressed on a cell (e.g., an endothelial cell associated with a blood brain barrier). In some embodiments, a targeting moiety which recognizes (e.g., binds to) a hTfR1 or a variant or a fragment thereof provides CNS-cell and / or tissue tropism. In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein bind to and / or recognize a target (e.g., hTfR1 or a variant or a fragment thereof) on a cell (e.g., an endothelial cell associated with a blood brain barrier). In some embodiments, binding of rAAV particles comprising a variant capsid having a peptide insertion disclosed herein to hTfR1 or a variant or a fragment thereof on a cell, delivers the rAAV particles to said cells. In some embodiments, rAAV particles delivered to cells by binding to hTfR1 or a variant or a fragment thereof allows transcytosis of said rAAV particles across said cell (e.g., to be transported from one surface of said cell to another surface of said cell) into an organ, an intersitial space, and / or an interstitial fluid allowing for transduction of a cell and / or tissue. In some embodiments, delivery of a variant AAV capsid protein to an organ, to an intersitial space, and / or to an interstitial fluid results in transduction of a cell or tissue. Also disclosed herein are compositions comprising rAAV particles disclosed herein, and uses of the same.

[0007] The present disclosure further encompasses peptides comprising CNS-targeting moieties (e.g., moieties targeting a CNS cell and / or tissue). Also disclosed herein are recombinant adeno-associated virus (rAAV) particles comprising a variant AAV capsid protein comprising a targeting moiety, e.g., a CNS-targeting moiety disclosed herein. In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety in a variant AAV capsid protein is also referred to as a “peptide insertion.” In some embodiments, a targeting moiety, e.g., a Page 2 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) CNS-targeting moiety, (e.g., in a variant AAV capsid protein), provides CNS-cell and / or tissue tropism. In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein bind to and / or recognize a target on a cell, e.g., a CNS cell and / or a cell in a CNS tissue, e.g., an endothelial cell associated with a blood brain barrier.

[0008] Without wishing to be bound by any particular theory, in some embodiments, variant AAV capsid proteins disclosed herein are particularly useful for targeting CNS cells and / or tissue expressing human Transferrin receptor (hTfR1) or a variant or a fragment thereof. In some embodiments, a variant AAV capsid protein disclosed herein binds to, e.g., specifically binds, to hTfR1 or a variant or a fragment thereof.

[0009] Further without wishing to be bound by any particular theory, in some embodiments, variant AAV capsid proteins disclosed herein can traverse a blood brain barrier (e.g., in a human subject) by binding to hTfR1 or a variant or a fragment thereof. In some embodiments, CNS cell and / or tissue tropism of a variant AAV capsid protein disclosed herein is a result of binding to hTfR1 or a variant or a fragment thereof.

[0010] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; (IV) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 Page 3 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

[0011] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; (IV) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

[0012] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 1. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 46 (RGADVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a Page 4 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) sequence of SEQ ID NO: 466 (RTEDVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 496 (RTEEVYP).

[0013] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 1. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 46 (RGADVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 466 (RTEDVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 496 (RTEEVYP).

[0014] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 2. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 80 (VNDYVSY).

[0015] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental Page 5 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 2. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 80 (VNDYVSY).

[0016] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 3. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 93 (WPNGVPG). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 91 (WDNGVPG).

[0017] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 3. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 93 (WPNGVPG). In some embodiments, Page 6 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 91 (WDNGVPG).

[0018] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 4. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 63 (RWDGADV).

[0019] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 4. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 63 (RWDGADV).

[0020] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a Page 7 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 5. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 396 (VSEFLTF).

[0021] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 5. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 396 (VSEFLTF).

[0022] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 6. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 483 (REDHVAW).

[0023] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a Page 8 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 6. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 483 (REDHVAW).

[0024] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 7. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 444 (AQALMHV).

[0025] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 7. In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 444 (AQALMHV).

[0026] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 Page 9 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 8.

[0027] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 8.

[0028] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of: YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 9.

[0029] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence of: YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 9.

[0030] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence provided in Table 10, Page 10 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) Table 11, Table 12, Table 13 or Table 14; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises a sequence provided in Table 10. In some embodiments, a peptide insertion comprises a sequence provided in Table 11. In some embodiments, a peptide insertion comprises a sequence provided in Table 12. In some embodiments, a peptide insertion comprises a sequence provided in Table 13. In some embodiments, a peptide insertion comprises a sequence provided in Table 14.

[0031] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of a sequence provided in Table 10, Table 11, Table 12, Table 13 or Table 14; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion consists of a sequence provided in Table 10. In some embodiments, a peptide insertion consists of a sequence provided in Table 11. In some embodiments, a peptide insertion consists of a sequence provided in Table 12. In some embodiments, a peptide insertion consists of a sequence provided in Table 13. In some embodiments, a peptide insertion consists of a sequence provided in Table 14.

[0032] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13 or Table 14; and (ii) a peptide insertion site is in a variable region of the parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 1. In Page 11 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 2. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence a sequence provided in Table 3. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 4. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 5. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 6. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 7. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 8. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 9. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 10. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 11. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 12. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 13. In some embodiments, a peptide insertion comprises at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in Table 14.

[0033] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion site is located between two adjacent amino acids in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0034] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion site is located between two non-adjacent amino acids in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0035] In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 Page 12 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVhu68 capsid protein, or an AAVrh10 capsid protein.

[0036] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0037] In some embodiments, a peptide insertion is in VR-VIII of a parental AAV capsid protein (e.g., an AAV9 capsid protein). In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10 capsid protein, and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein or the corresponding positions in a capsid protein (e.g., a VP1, VP2 or VP3) of another parental AAV capsid protein e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10 capsid protein.

[0038] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0039] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 1, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0040] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 2, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0041] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion Page 13 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) comprising a consensus sequence of SEQ ID NO: 3, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0042] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 4, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0043] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 961, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0044] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 962, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0045] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 963, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0046] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 964, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0047] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of SEQ ID NO: 965, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0048] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion Page 14 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) comprising a sequence provided in Table 10, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0049] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a sequence provided in Table 11, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0050] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a sequence provided in Table 12, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0051] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a sequence provided in Table 13, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0052] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a sequence provided in Table 14, and (2) one or more sequences of a VP, e.g., a VP1, VP2, or VP3, of an AAV9 capsid protein.

[0053] In some embodiments, a peptide insertion is in VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0054] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 46 (RGADVYP) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 46 (RGADVYP) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0055] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 466 (RTEDVYP) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: Page 15 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 466 (RTEDVYP) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0056] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 496 (RTEEVYP) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 496 (RTEEVYP) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0057] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 80 (VNDYVSY)and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 80 (VNDYVSY) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0058] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 93 (WPNGVPG) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 93 (WPNGVPG) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0059] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 91 (WDNGVPG) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 91 (WDNGVPG) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0060] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 63 (RWDGADV) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 63 (RWDGADV) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0061] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 396 (VSEFLTF) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an Page 16 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 396 (VSEFLTF) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0062] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 483 (REDHVAW) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 483 (REDHVAW) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0063] In some embodiments, a peptide insertion comprises a sequence of SEQ ID NO: 444 (AQALMHV) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is a sequence of SEQ ID NO: 444 (AQALMHV) and is inserted between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0064] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0065] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0066] This disclosure further provides a targeting moiety, e.g., a CNS-targeting moiety, conjugated or fused to a payload. In some embodiments, a targeting moiety, e.g., a CNS- targeting moiety, comprises a peptide sequence provided in any one of Tables 1-14. In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, comprises a peptide sequence comprising at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in provided in any one of Tables 1-14. In some embodiments, a targeting moiety, e.g., a CNS- targeting moiety, is part of (e.g., incorporated into) a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, encapsidates a payload, e.g., as described herein. In some embodiments, a targeting moiety, e.g., a CNS- Page 17 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) targeting moiety, is tethered to a payload, e.g., as described herein. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 46 (RGADVYP). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 93 (WPNGVPG). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 91 (WDNGVPG). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 63 (RWDGADV). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 466 (RTEDVYP). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 496 (RTEEVYP). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 80 (VNDYVSY). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 396 (VSEFLTF). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 483 (REDHVAW). In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 444 (AQALMHV).

[0067] Also disclosed herein is an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein or a targeting moiety, e.g., a CNS- targeting moiety, fused to a payload.

[0068] Further disclosed herein is an isolated cell transduced with an rAAV particle disclosed herein. Also disclosed herein is a cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein.

[0069] This disclosure also provides a composition comprising a targeting moiety, e.g., a CNS-targeting moiety, and a payload, wherein the targeting moiety, e.g., a CNS-targeting moiety, comprises a peptide sequence provided in any one of Tables 1-14. In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, comprises a peptide sequence comprising at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in provided in any one of Tables 1-14.

[0070] Further provided herein is a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid Page 18 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) protein, and wherein: (i) a peptide insertion comprises a sequence of: (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; (IV) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, and (ii) a peptide insertion site is in a variable region (VR) of the parental AAV capsid protein (e.g., of an AAV9 capsid protein).

[0071] This disclosure provides a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) a peptide insertion comprise a sequence provided in any one of Tables 1-14; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 46 (RGADVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 93 (WPNGVPG). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 91 (WDNGVPG). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 63 (RWDGADV). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 466 (RTEDVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 496 (RTEEVYP). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 80 (VNDYVSY). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 396 (VSEFLTF). In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 483 (REDHVAW). In some Page 19 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 444 (AQALMHV).

[0072] This disclosure provides a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) a peptide insertion comprise a sequence of at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13 or Table 14; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0073] In some embodiments, a peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR- V, VR-VI, VR-VII, VR-VIII or VR-IX of a parental AAV capsid protein (e.g., an AAV9 capsid protein). In some embodiments, a peptide insertion is in VR-VIII of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0074] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and the VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0075] Also provided herein is a composition comprising a targeting moiety, e.g., a CNS- targeting moiety, and a payload, wherein a targeting moiety, e.g., a CNS-targeting moiety, comprises: a sequence of: (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; (IV) RX1DGADV (SEQ ID NO:4) wherein X1is Y, W, or F; (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A. Page 20 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0076] Also provided herein is a composition comprising a targeting moiety, e.g., a CNS- targeting moiety, and a payload, wherein a targeting moiety, e.g., a CNS-targeting moiety, comprises a peptide comprising a sequence provided in any one of Tables 1-14. In some embodiments, a peptide insertion in a targeting moiety, e.g., a CNS-targeting moiety, comprises a sequence of: SEQ ID NO: 46 (RGADVYP), SEQ ID NO: 93 (WPNGVPG), SEQ ID NO: 91 (WDNGVPG), SEQ ID NO: 63 (RWDGADV), SEQ ID NO: 466 (RTEDVYP), SEQ ID NO: 496 (RTEEVYP), SEQ ID NO: 80 (VNDYVSY), SEQ ID NO: 396 (VSEFLTF), SEQ ID NO: 483 (REDHVAW), or SEQ ID NO: 444 (AQALMHV).

[0077] Further provided herein is a composition comprising a targeting moiety, e.g., a CNS-targeting moiety, and a payload, wherein a targeting moiety, e.g., a CNS-targeting moiety, comprises a peptide comprising a sequence of at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, or Table 14.

[0078] In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, is conjugated or fused to a payload.

[0079] In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, is inserted into a viral protein, e.g., an AAV capsid protein.

[0080] In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, is part of, e.g., incorporated into, a vector.

[0081] In some embodiments, a targeting moiety, e.g., a CNS-targeting moiety, is not part of a vector.

[0082] This disclosure further provides a pharmaceutical composition comprising: (a) a rAAV particle disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0083] Also provided herein is a method of delivering a payload to a CNS cell and / or tissue, comprising administering a pharmaceutical composition disclosed herein to a CNS cell and / or tissue.

[0084] In some embodiments, a CNS cell and / or tissue is in vitro.

[0085] In some embodiments, a CNS cell and / or tissue is in vivo. Page 21 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0086] In some embodiments, a CNS cell and / or tissue is from a subject that has, or has been determined to have, a CNS disorder.

[0087] In some embodiments, a CNS cell or tissue is chosen from: a CNS epithelial cell, an endothelial cell associated with a blood brain barrier, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or a combination thereof.

[0088] In some embodiments, a CNS cell comprises an endothelial cell associated with a blood brain barrier. In some embodiments, an endothelial cell associated with a blood brain barrier is part of, or forms a blood brain barrier. In some embodiments, an endothelial cell associated with a blood brain barrier is or comprises a brain capillary endothelial cell (BCEC). In some embodiments, an endothelial cell associated with a blood brain barrier is or comprises a brain microvascular endothelial cell (BMEC). In some embodiments, an endothelial cell associated with a blood brain barrier expresses a hTfR1 or a fragment or variant thereof.

[0089] In some embodiments, a CNS cell comprises a CNS epithelial cell. In some embodiments, a CNS epithelial cell comprises a cell that lines one or more brain ventricles.

[0090] In some embodiments, a CNS cell comprises a nerve cell (neuron). In some embodiments, a neuron comprises a unipolar neuron, a bipolar neuron, a pseudounipolar neuron, a multipolar neuron, or combinations thereof. In some embodiments, a neuron is a motor neuron, a sensory neuron, an interneuron, an excitatory neuron, an inhibitor neuron, a sympathetic neuron, a parasympathetic neuron, or combinations thereof. In some embodiments, a neuron comprises a pyramidal neuron, a dopaminergic neuron, a cholinergic neurons, an adrenergic neuron, a GABAergic neuron, a glutamatergic neuron, a serotonergic neuron, a purinergic neuron, a histaminergic neuron, a lower motor neuron, or combinations thereof.

[0091] In some embodiments, a nerve cell (neuron) comprises nerve support cells. In some embodiments, nerve support cells comprise glial cells. In some embodiments, glial cells comprise astrocytes, microglial cells, ependymal cells, oligodendrocytes, Schwann cells, or combinations thereof. Page 22 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0092] In some embodiments, a CNS cell comprises a CNS connective tissue cell. In some embodiments, a CNS connective tissue cell comprises fat cells or meninges cells, or both.

[0093] In some embodiments, a CNS cell comprises a stem cell or progenitor cell. In some embodiments, a stem cell comprises a neural stem cell.

[0094] In some embodiments, a CNS cell comprises a cell that lines one or more brain ventricles.

[0095] In some embodiments, a CNS cell comprises a meninges cell.

[0096] In some embodiments, a CNS cell comprises a fat cell.

[0097] In some embodiments, a CNS tissue comprises tissue found in: cortex, thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, deep cerebellar nuclei, or other parts of the brain and / or spinal cord.

[0098] In some embodiments, CNS tissue comprises tissue found in a frontal cortex, a parietal cortex, an occipital cortex, a temporal cortex or combinations thereof.

[0099] In some embodiments of any of the AAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a variant AAV capsid protein confers increased infectivity and / or transduction of a central nervous system (CNS) cell and / or tissue compared to the infectivity and / or transduction of the CNS cell and / or tissue by a control AAV particle comprising a corresponding parental AAV capsid protein.

[0100] In some embodiments, a rAAV particle comprising a variant AAV capsid protein confers at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 120-fold, at least 140- fold, at least 160-fold, at least 180-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900- fold, at least 1000-fold, at least 1500-fold, at least 2000-fold, at least 4000-fold increased infectivity and / or transduction of a CNS cell compared to: (1) infectivity and / or transduction of a CNS cell by a control AAV particle comprising a corresponding parental AAV capsid protein; or Page 23 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) (2) infectivity and / or transduction of a CNS cell which does not express a receptor recognized by a variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

[0101] In some embodiments, a rAAV particle comprising a variant AAV capsid protein confers about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20- fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 120-fold, about 140-fold, about 160-fold, about 180-fold, about 200-fold, about 250-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1500- fold, about 2000-fold, about 4000-fold, increased infectivity and / or transduction of a CNS cell or tissue compared to: (1) infectivity and / or transduction of a CNS cell by a control AAV particle comprising a corresponding parental AAV capsid protein; or (2) infectivity and / or transduction of a CNS cell which does not express a receptor recognized by a variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

[0102] In some embodiments, a rAAV particle comprising a variant AAV capsid protein confers 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15- fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120- fold, 140-fold, 160-fold, 180-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, 4000-fold, increased infectivity and / or transduction of a CNS cell or tissue compared to: (1) infectivity and / or transduction of a CNS cell by a control AAV particle comprising a corresponding parental AAV capsid protein; or (2) infectivity and / or transduction of a CNS cell which does not express a receptor recognized by a variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

[0103] In some embodiments of any of the variant AAV capsid proteins, AAV particles comprising a variant AAV capsid protein, compositions comprising a variant AAV capsid protein, or methods of using the same, a variant AAV capsid protein comprises one or more modifications to an amino acid sequence flanking a peptide insertion. In some embodiments, one or more modifications are within about 10 amino acids upstream or downstream of the location of a peptide insertion site, e.g., within about 5 amino acids upstream or downstream of the location of the peptide insertion site. Page 24 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0104] In some embodiments, one or more modifications comprises an insertion, deletion, mutation, or combinations thereof.

[0105] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications are located in a variable region of parental AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, one or more modifications are located in an AAV9 capsid protein variable region, e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX, or any combination thereof.

[0106] In some embodiments, one or more modifications are located in: VR-VIII of a VP1, VP2, or VP3 of an AAV9 capsid protein.

[0107] In some embodiments, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose. By way of example, glycan binding residues of an AAV9 capsid protein include, but are not limited to: (a) amino acids 271 and 272 of a VP1, VP2 or VP3; (b) amino acid 446 of a VP1, VP2 or VP3; (c) amino acid 470 of a VP1, VP2 or VP3; (d) amino acids 501 to 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3; or (e) amino acids 489 and 545 of a VP1, VP2 or VP3. Based on the exemplary glycan binding domains and / or residues provided herein for an AAV9 capsid protein, those with knowledge in the pertinent field would be able to readily ascertain the corresponding glycan binding domain and / or residues in a different parental AAV capsid protein.

[0108] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications is at a glycan binding domain of a parental AAV capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10 capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein.

[0109] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an Page 25 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0110] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (b) 446 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (c) 470 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3 of an AAV9 capsid protein; (e) 489 and 545 of VP1of a VP1, VP2 or VP3 of an AAV9 capsid protein; (f) 591 and 621 of a VP1, VP2 or VP3 of an AAV9 capsid protein; or (g) any combination or all of (a)-(f).

[0111] In some embodiments of any of the variant AAV capsid proteins, AAV particles comprising a variant AAV capsid protein, compositions comprising a variant AAV capsid protein, or methods of using the same, a variant AAV capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity relative to a parental AAV capsid protein.

[0112] In some embodiments, percent identity is determined by comparing the sequence of a variant AAV capsid protein without a peptide insertion, with a parental AAV capsid protein.

[0113] In some embodiments, a variant AAV capsid protein and a parental AAV capsid protein have 100% identity when: (a) a peptide insertion in a variant AAV capsid protein is not taken into account in the sequence comparison; and (b) a variant AAV capsid protein does not have one or more modifications other than a peptide insertion.

[0114] In some embodiments, a variant AAV capsid protein and a parental AAV capsid protein have less than 100% identity when: (a) a peptide insertion in a variant AAV capsid Page 26 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) protein is not taken into account in the sequence comparison; and (b) a variant AAV capsid protein comprises one or more modifications other than a peptide insertion.

[0115] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001.

[0116] In some embodiments, a parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002.

[0117] In some embodiments, a parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003.

[0118] In some embodiments, a parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2010.

[0119] In some embodiments, a parental AAV capsid protein is an AAV4 capsid protein of SEQ ID NO: 2051.

[0120] In some embodiments, a parental AAV capsid protein is an AAV5 capsid protein of SEQ ID NO: 2004.

[0121] In some embodiments, a parental AAV capsid protein is an AAV6 capsid protein of SEQ ID NO: 2005.

[0122] In some embodiments, a parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006.

[0123] In some embodiments, a parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2052.

[0124] In some embodiments, a parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006.

[0125] In some embodiments, a parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2053.

[0126] In some embodiments, a parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2054. Page 27 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0127] In some embodiments, a parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2055.

[0128] In some embodiments, a parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2056.

[0129] In some embodiments, a parental AAV capsid protein is an AAVhu68 capsid protein of SEQ ID NO: 2057.

[0130] In some embodiments, a parental AAV capsid protein is an AAVrh10 capsid protein of SEQ ID NO: 2058.

[0131] In some embodiments of any of the AAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a payload is or comprises a polypeptide that is encoded by a nucleic acid sequence within a rAAV particle.

[0132] In some embodiments, a polypeptide is or comprises a CRISPR-Cas protein, or a variant or fragment thereof. In some embodiments, a CRISPR-Cas protein is chosen from : a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment of any of the foregoing. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA / tracrNA that interacts with the CRISPR-Cas protein. In some embodiments a CRISPR-Cas protein is fused with one or more domains, e.g., an activator domain and / or a repressor domain. In some embodiments a CRISPR-Cas protein is a nuclease. In some embodiment a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleic acid molecule. In some embodiment a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule.

[0133] In some embodiments, a polypeptide is or comprises a Zinc finger protein, or a variant or fragment thereof. In some embodiments, a Zinc finger protein is chosen from: a Zinc finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeted zinc-finger DNA-binding unit optionally fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or any combination of any of the foregoing. In some embodiments a Zinc Page 28 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) figner protein is fused with one or more domains, e.g., an activator domain and / or a repressor domain.

[0134] In some embodiments, a polypeptide is or comprises a Transcription Activator- Like Effector (TAL) protein, or a variant or fragment thereof. In some embodiments, a TAL comprises: a TAL effector DNA binding domain (e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.), a Transcription Activator-Like Effector Nuclease (TALEN), e.g., a TAL effector DNA binding domain fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or any combination of any of the foregoing. In some embodiments a TAL protein is fused with one or more domains, e.g., an activator domain and / or a repressor domain.

[0135] In some embodiments, a polypeptide is or comprises a base editor, or a variant or fragment thereof. In some embodiments, a base editor comprises a deaminase, an adenosine deaminase enzyme (ABE), a cytosine deaminase enzyme (CBE), an APOBEC1, an APOBEC3A, an APOBEC3G, an evoAPOBEC, a BE4-YE1, a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine deaminases (TadA*), an E. coli tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA binding domain monomer, a base editing enzyme that is RNA guided, a DNA glyosylase inhibor, one or more DNA glycosylase inhibitor domains, a 5-methylcytosine deaminase, a cytidine deaminase domain, an adenine deaminase domain, an adenosine base editor (ABE), a Target-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax., a circularly permuted base editor, an adenosine deaminase enzyme (ADAR), a RNA editing for programmable adenosine to inosine replacement (REPAIR), a leveraging endogenous ADAR for programmable editing of RNA (LEAPER) or a variant or fragment or combination of any of the foregoing. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA / tracrNA that interacts with the base editor.

[0136] In some embodiments, a polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising the same. In some embodiments, a prime editor and / or system comprising the same comprises: a reverse transcriptase, a prime editing enzyme, an editing enzyme that includes a reverse transcriptase domain, an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Murine Leukemia Virus (MLV) Reverse Transcriptase, a HIV- Page 29 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 1 reverse transcriptase, a bacterial reverse transcriptase, a reverse transcriptase associated with a DNA binding domain and / or protein, a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase), a prime editing 1 system (PE1), a prime editing 2 system (PE2), a prime editing 3 system (PE3), a prime editing 3b system (PE3b) or a variant or fragment or any combination of any of the foregoing. In some embodiments, the payload also comprises a prime editing gRNA (pegRNA) or an extended sgRNA that interacts with the prime editor.

[0137] In some embodiments, a polypeptide is or comprises a meganuclease, or a variant or fragment thereof. In some embodiments, a meganuclease is chosen from: a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His- Cyst box family meganuclease, or HNH family endonuclease, an I-SeeI, an I-CeuI, a PI-PspI, a PI-SceI, an I-SceIV, an I-CsmI, an I-PanI, an I-SceII, an I-PpoI, an I-SceIII, an I-CreI, an I-TevI, an I-TevII an I-TevIII or a variant or fragment or any combinations of any of the foregoing.

[0138] In some embodiments, a polypeptide is associated with a CNS disorder.

[0139] In some embodiments, a CNS disorder is a result of a genetic abnormality.

[0140] In some embodiments, a CNS disorder is a not a result of a genetic abnormality.

[0141] In some embodiments, a CNS disorder is chosen from: Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay-Sachs, Spinal Muscular Atrophy, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies (e.g., a STXBP1 genetic epilepsy, or a CDKL5 genetic epilepsy), or combinations thereof.

[0142] In some embodiments, a polypeptide is an enzyme.

[0143] In some embodiments, a polypeptide is an antibody.

[0144] In some embodiments, a polypeptide is a secreted protein.

[0145] In some embodiments of any of the rAAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same Page 30 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) disclosed herein, a payload is or comprises an RNA molecule that is encoded by a nucleic acid sequence within a rAAV particle. In some embodiments, an RNA molecule is or comprises an siRNA, a miRNA, a gRNA, an antisense RNA, a circular RNA, an snRNA or an aptamer.

[0146] In some embodiments, an RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a CNS disorder.

[0147] In some embodiments of any of the rAAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a payload is or comprises a DNA molecule. In some embodiments, a DNA molecule is or comprises a nucleic acid sequence of up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc. In some embodiments, a DNA molecule is or comprises a nucleic acid sequence of up to 5,100 nt in length, e.g., up to 5,000 nt, up to 4,900, up to 4,800, up to 4,700, up to 4,600, up to 4,500, up to 4,400, etc.

[0148] In some embodiments of any of the rAAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a nucleotide sequence encoding a payload is operably linked to a promoter.

[0149] In some embodiments, a promoter is a CNS promoter or a variant or a fragment thereof. In some embodiments, a CNS promoter is chosen from: a GFAP promoter, a SYN1 promoter, a NSE / RU5’ promoter, a neuroactive peptide cholecystokinin (CCK) promoter, a myelin basic promoter (MBP), a human myelin associated glycoprotein promoter, a phosphate- activated glutaminase (PAG) promoter, a vesicular glutamate transporter (vGLUT) promoter, a glutamic acid decarboxylase (GAD) promoter, Camk2a promoter, TH (tyrosine hydroxylase) promoter, Hb9 promoter, CNP promoter, NES (nestin) promoter, Tub1a promoter, SST (somatostatin) promoter, MeCP2 promoter, or combinations thereof.

[0150] In some embodiments, a promoter is or comprises a chicken beta actin hybrid (CBh) promoter or a variant or a fragment thereof.

[0151] In some embodiments, a promoter is or comprises a hSyn1 promoter or a variant or a fragment thereof. Page 31 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0152] In some embodiments, a promoter is or comprises a GFAP promoter or a variant or a fragment thereof.

[0153] In some embodiments of any of the methods of using a pharmaceutical composition comprising a rAAV particle comprising a variant AAV capsid protein disclosed herein, a pharmaceutical composition is administered via a route of administration chosen from: intravenous, intraarterial, intracoronary, intraparenchymal, subpial, intrathecal, intraocular, intracerebroventricular (ICV), intracisternal magna (ICM), or intramuscular.

[0154] In some embodiments, a subject is a human.

[0155] Also provided herein is a method of treating a subject having a CNS disorder and / or ameliorating a symptom of a CNS disorder in a subject, the method comprising administering to the subject a pharmaceutical composition disclosed herein.

[0156] In some embodiments, a CNS disorder is chosen from Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay- Sachs, Spinal Muscular Atrophy, Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies (e.g., a STXBP1 genetic epilepsy, or a CDKL5 genetic epilepsy), or combinations thereof.

[0157] As would be understood by one with ordinary skill in the art, a peptide sequence disclosed herein, e.g., a peptide disclosed in any one of Tables 1-14, can be used as a targeting moiety, e.g., a CNS targeting moiety, to deliver a payload. In some embodiments, a targeting moiety, e.g., a CNS targeting moiety, can be conjugated or fused to a payload. In some embodiments, a targeting moiety, e.g., a CNS targeting moiety, is inserted in a viral protein, e.g., an AAV capsid.

[0158] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. Page 32 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) BRIEF DESCRIPTION OF THE DRAWING

[0159] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0160] The Figures described below, which together make up the Drawing, are for illustration purposes only, not for limitation

[0161] FIGs.1A-1B are dot plots showing results from a Round 2 in vivo selection to identify variant AAV capsid proteins with an insertion at VR-VIII of AAV9 that show enhanced CNS transduction in human Transferrin Receptor (hTfR1) knock-in (hTfR1-KI)mice compared to WT mice. Each dot represents a capsid variant. The x-and y-axes correspond to Apparent Enrichment (Log2) of capsid variants in WT and hTfR1-KI mouse brain, respectively. Blue dots in FIG.1A represent variants with over 10 fold greater Apparent Enrichment in hTfR1-KI brains versus WT brains (FDR < 0.1). Colored dots in FIG.1B correspond to hTfR1-dependent capsid candidates with distinct peptide motifs.

[0162] FIGs.2A-2B show the results of a characterization and confirmation screen using a small number of capsid variants selected from the Round 2 selection with barcodes driven by a CAG or human synapsin 1 (hSyn-1) gene promoter. Each symbol represents a capsid variant. Filled circle is benchmark AAV9. Star represents mouse CNS-targeting capsid carrying the NGNPGRW (SEQ ID NO: 3016) peptide insertion which contains a LY6C1 interacting motif, as a control. Filled squares are capsids with motif RxxxxYP (SEQ ID NO: 3011). Asterisk is capsid with motif xNDYVSY (SEQ ID NO: 2). Black up-pointing triangles are capsids with motif [W / Y]xNG[V / N][P / R]G (SEQ ID NO: 3013). Black down-pointing triangle is capsid with motif R[W / Y]DGADV (SEQ ID NO: 3014). Open circles are unique sequences without the indicated motifs. Crosses are non-hits. FIG.2A compares CNS transduction results between variant AAV capsid proteins with barcodes driven by CAG and hSYN-1 promoters. FIG.2B compares transduction performance of AAV capsid variants in brain, liver, heart, and quadriceps of hTfR1- KI and WT mice.

[0163] FIG.3 shows the results of an in vitro confirmation screen using barcoded AAV capsid variants in TfR1-knockout or hTfR1-expressing CHO cells. Each symbol represents a Page 33 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid variant. Filled circle is benchmark AAV9. Star represents mouse CNS-targeting capsid carrying the NGNPGRW (SEQ ID NO: 3016) peptide insertion which contains a LY6C1 interacting motif, as a control. Filled squares are capsids with motif RxxxxYP (SEQ ID NO: 3011). Asterisk is capsid with motif xNDYVSY (SEQ ID NO: 2). Black up-pointing triangles are capsids with motif [W / Y]xNG[V / N][P / R]G (SEQ ID NO: 3013). Black down-pointing triangle is capsid with motif R[W / Y]DGADV (SEQ ID NO: 3014). Open circles are unique sequences without the indicated motifs. Crosses are non-hits. Black colors and gray colors represent measurements in the absence and presence of human transferrin (Tf), respectively, where measurements for the same capsid are connected by a line segment.

[0164] FIGs.4A-4B show the results of an assessment of vector transduction in hTfR1- KI mice brain, spinal cord, quadriceps, and liver of certain AAV capsid hits identified in Round 2 selection. The animals were divided into 6 groups: “1” corresponds to AAV9 particles in hTfR1-KI mice; “2” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) peptide inserted between positions 588 and 589 of VP1 in hTfR1-KI mice; “3” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1 in hTfR1-KI mice; “4” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RWDGADV (SEQ ID NO: 63) peptide inserted between positions 588 and 589 of VP1 in hTfR1-KI mice; “5” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RGADVYP (SEQ ID NO: 46) peptide inserted between positions 588 and 589 of VP1 in hTfR1-KI mice; and “6” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RGADVYP (SEQ ID NO: 46) peptide inserted between positions 588 and 589 of VP1 in WT mice. Each dot represents a biological replicate; closed circles and open squares represent male and female mice, respectively. Open bars represent normalized mCherry mRNA fold enhancement over Group 1 (shown as “1”); shaded bars represent vector genome copies per cell. FIG.4A shows variant AAV capsid protein transduction in each respective organ as measured by CAG-driven mCherry transgene mRNA expression. FIG.4B shows variant AAV capsid protein biodistribution in each respective organ as measured by vector genome per diploid genome. P values: * indicates a p-value between 0.01 and 0.05, ** indicates a p-value between 0.001 and 0.01; *** indicates a p-value between 0.0001 and 0.001, and **** indicates a p-value of less than 0.0001. Page 34 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0165] FIGs.5A-5B are a panel of images comparing the results of CNS transduction, as observed by immunohistochemistry (IHC), of various brain regions of hTfR1-KI mice treated with AAV9 particles or an AAV particle comprising an exemplary variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) or WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1. The top panel shows mCherry IHC images of brain section from hTfR1-KI mice treated with AAV9 particles. Middle panel shows mCherry IHC images of brain section from hTfR1-KI mice treated with AAV particles comprising exemplary variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) peptide inserted between positions 588 and 589 of VP1. Bottom panel shows mCherry IHC images of brain section from hTfR1-KI mice treated with AAV particles comprising exemplary variant AAV9 capsid protein with the WDNGVPG(SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1. For this animal, high magnification images of the cortical regions were not available. FIG.5A is in color and FIG.5B is in black and white.

[0166] FIG.6 show the results of an assessment of AAV transduction in brain, spinal cord, quadriceps, and liver of AAV9 particles or four certain AAV capsid hits identified in Round 2 selection in B-hTFR1 mice. “1” corresponds to an AAV particle comprising an AAV9 capsid in B-hTFR1 mice; “2” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “3” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “4” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RWDGADV (SEQ ID NO: 63) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; and “5” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RGADVYP (SEQ ID NO: 46) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice.

[0167] All male animals were used in this study. Each dot represents a biological replicate. Open bars represent normalized mCherry mRNA fold enhancement over Group 1. Group 1 was dosed at 5E+13 vg / kg, while Group 2, 3, 4 were dosed at 2E+13 vg / kg.

[0168] FIGs.7A-7B are a panel of images comparing the results of CNS transduction, as observed by immunohistochemistry (IHC), of various brain regions of B-hTFR1 mice treated Page 35 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) with AAV9 particles or an AAV particle comprising an exemplary variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) or WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1. The top panel shows mCherry IHC images of brain section from B-hTFR1 mice treated with AAV9 particles. Middle panel shows mCherry IHC images of brain section from B-hTFR1 mice treated with AAV particles comprising exemplary variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) peptide inserted between positions 588 and 589 of VP1. Bottom panel shows mCherry IHC images of brain section from B-hTFR1 mice treated with AAV particles comprising exemplary variant AAV9 capsid protein with the WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1. FIG.7A is in color and FIG.7B is in black and white.

[0169] FIGS.8A-8C are a panel of graphs showing optimization library screening in hTfR1-KI mice and WT mice. Each dot represents a capsid variant. Blue dots in the top right quadrant of FIG.8A represent variants with over 10-fold enhanced CNS enhancement over AAV9 with at least a 2-fold enhancement of brain transduction in B-hTFR1 mice compared to hTfR1 KI- / -mice (FDR < 0.1). Colored dots marked by labels in FIG.8B and 8C represent top hTfR1-dependent capsids that were characterized in vivo.

[0170] FIG.9 is a graph depicting results from the evaluation of receptor-mediated transduction for exemplary capsid candidates. Each novel capsid, packaging a firefly luciferase transgene driven by a CAG promoter, was produced by HEK293 cells in a 6-well plate. Crude cell lysate was collected and directly applied to parental CHO cells with endogenous TfR1 knockout (KO) or cells stably expressing cynoTfR1 or hTfR1 at three different dilutions. To mimic the in vivo environment, 10 μM transferrin (Tf) was added to the cell culture during viral transduction. Luciferase signals were measured after 48 hours, and data analysis was performed to calculate the cynoTfR1- or hTfR1-mediated transduction compared to AAV9.

[0171] FIGS.10A-10B are graphs showing AAV transduction or vector genomes in brain, spinal cord, quadriceps or liver in animals administered AAV9 particles or AAV particles comprising exemplary variant AAV9 capsid proteins with certain peptides inserted between position 588 and 589 of VP1 or AAV9 particles in a hTfR1-KI mouse model (B-hTFR1). In FIG.10A, “1” and “2” correspond to an AAV particle comprising an AAV9 capsid; “3” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WDNGVPG Page 36 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice, “4” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RTEDVYP (SEQ ID NO: 466) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “5” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the REDHVAW (SEQ ID NO: 483) peptide inserted between positions 588 and 589 of VP1 in B- hTFR1 mice; and “6” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the VSEFLTF (SEQ ID NO: 396) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice. In FIG.10B, “1” corresponds to an AAV particle comprising an AAV9 capsid; “2” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WPNGVPG (SEQ ID NO: 93) peptide inserted between positions 588 and 589 of VP1 in B- hTFR1 mice; “3” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “4” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RWDGADV (SEQ ID NO: 63) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “5” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RGADVYP (SEQ ID NO: 46) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “6” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RTEDVYP (SEQ ID NO: 466) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; “7” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the REDHVAW (SEQ ID NO: 483) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice; and “8” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the VSEFLTF (SEQ ID NO: 396) peptide inserted between positions 588 and 589 of VP1 in B-hTFR1 mice. All male animals were used in this study. All groups, except for group 2 shown in FIG.10A, were dosed at 2E+13 vg / kg. Group 2 shown in FIG.10A was dosed at 5E+13 vg / kg. Each dot represents a biological replicate. Open bars represent the normalized fold enhancement of mCherry mRNA relative to the brain transduction level of Group 1; shaded bars represent vector genome copies per cell.

[0172] FIGs.11A-11B are a panel of images comparing the results of CNS transduction, as observed by immunohistochemistry (IHC), of various brain regions of hTfR1-KI mice (B- hTFR1) treated with AAV9 particles or an AAV particle comprising an exemplary variant AAV9 capsid protein with the RTEDVYP (SEQ ID NO: 466), REDHVAW (SEQ ID NO: 483), Page 37 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) or VSEFLTF (SEQ ID NO: 396) peptide inserted between positions 588 and 589 of VP1. The top panel shows mCherry IHC images of brain section from hTfR1-KI mice treated with AAV9 particles. Second, third and fourth panels show mCherry IHC images of brain section from hTfR1-KI mice treated with AAV particles comprising exemplary variant AAV9 capsid protein with the RTEDVYP (SEQ ID NO: 466), REDHVAW (SEQ ID NO: 483), or VSEFLTF (SEQ ID NO: 396) peptides respectively inserted between positions 588 and 589 of VP1. FIG.11A is in color and FIG.11B is in black and white.

[0173] FIG.12 is a graph depicting AAV transduction in brain, spinal cord, quadriceps or liver in animals administered AAV9 particles or AAV particles comprising exemplary variant AAV9 capsid proteins with certain peptides inserted between position 588 and 589 of VP1 or AAV9 particles in in WT C57BL / 6 mice. “1” corresponds to an AAV particle comprising an AAV9 capsid; “2” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the RTEDVYP (SEQ ID NO: 466) peptide inserted between positions 588 and 589 of VP1 in WT C57BL / 6 mice; “3” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the REDHVAW (SEQ ID NO: 483) peptide inserted between positions 588 and 589 of VP1 in WT C57BL / 6 mice; “4” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the VSEFLTF (SEQ ID NO: 396) peptide inserted between positions 588 and 589 of VP1 in WT C57BL / 6 mice; and “5” corresponds to an AAV particle comprising a variant AAV9 capsid protein with the WDNGVPG (SEQ ID NO: 91) peptide inserted between positions 588 and 589 of VP1 in WT C57BL / 6 mice. Each dot represents a biological replicate. All male animals were used in this study. All 5 groups were dosed at 2E+13 vg / kg. Open bars represent the normalized fold enhancement of mCherry mRNA relative to the brain transduction level of Group 1.

[0174] FIG.13 shows upstream productivity of AAV particles comprising exemplary variant AAV9 capsid proteins with certain peptides inserted between position 588 and 589 of VP1. Upstream production was compared to AAV9 particles. Two different cargos were packaged into the corresponding capsids for productivity assessment, as listed in the figure.

[0175] FIG.14 shows results from a Bio-Layer Interferometry (BLI) binding evaluation of selected capsids to TfR1. Recombinant proteins of human TfR1 or cynoTfR1 extracellular domains were captured at a concentration of 50nM on Octet HIS1K biosensors. Capsids at 1e11 Page 38 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) GC / mL were associated for 1hr then dissociated in buffer for 1hr. Binding interactions between capsids and recombinant TfR1 were monitored using the Octet HTX Instrument. DEFINITIONS

[0176] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0177] 5’ and 3’: The terms “5’” and “3’” are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence. Thus, 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment, while 5’ indicates a segment of the nucleic acid that is upstream of another segment. For example, 3’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence. Similarly, 5’ may indicate that a segment is in the 5’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence. Unless indicated otherwise, the directionality of a nucleic acid will be in the 5’ to 3’ direction of translation.

[0178] About or approximately: As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0179] Adeno-associated virus (AAV): As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in whole or in part, of the family Parvoviridae and the genus Dependoparvovirus. AAV is a small replication-defective, nonenveloped virus. AAV includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, Page 39 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, non-human primate AAV, e.g., from rhesus monkeys, and any variant of any of the foregoing. Wild-type AAV is replication deficient and requires co-infection of cells by a helper virus, e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate.

[0180] Ad2 helper: As used herein, the term “Ad2 helper” refers to the Adenovirus serotype 2 (Ad2) helper virus (e.g., wildtype or recombinantly engineered Ad2 helper virus) and various Ad2 helper genes and / or Ad2 helper polypeptides or nucleic acids, including, but not limited, to E1a, E1b, E2a, E4Orf6, VA RNA, and any variant or fragment of any of the foregoing. In some embodiments, an Ad2 helper vector (e.g., plasmid) encodes Ad2 helper polypeptides or nucleic acids (e.g., one, two, three, or four of E1 (e.g., E1a and / or E1b), E2a, E4, or VA RNA) necessary to generate functional rAAV particles. In certain embodiments, the Ad2 helper vector is transfected into an E1 complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad2 helper vector and Ad2 helper virus genes can be derived from the Adenovirus 2 genome (GenBank Accession No. J01917.1).

[0181] Ad5 helper: As used herein, the term “Ad5 helper” refers to the Adenovirus serotype 5 (Ad5) helper virus (e.g., wildtype or recombinantly engineered Ad5 helper virus) and various Ad5 helper genes and / or Ad5 helper polypeptides or nucleic acids, including, but not limited, to E1a, E1b, E2a, E4Orf6, and / or VA RNA. In some embodiments, an Ad5 helper vector (e.g., plasmid) encodes Ad5 helper polypeptides or nucleic acids (e.g., one, two, three, or four of E1 (e.g., E1a and / or E1b), E2a, E4, or VA RNA) necessary to generation functional rAAV particles. In certain embodiments, the Ad5 helper vector is transfected into an E1 complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad5 helper vector and Ad5 helper genes can be derived from the Adenovirus 5 genome (GenBank Accession No. AY601635).

[0182] Administration: As used herein, the term “administration” refers to the administration of a composition comprising rAAV particles as described herein to a subject. Administration may be by any appropriate route. For example, in some embodiments, administration may be local or systemic administration (e.g., to a mammal, e.g., to a human, e.g., a patient). A composition of the disclosure may be administered by injection or infusion by any Page 40 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) route. For example, a composition may be administered by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intracisternal magna, or intrathecal injection or infusion. Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal.

[0183] Bioreactor: The term “bioreactor,” as used herein, refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). The bioreactor can be of any size and / or any shape so long as it is useful for culturing a cell culture (e.g., a mammalian cell culture).

[0184] Cap polypeptide: As used herein, the term “Cap polypeptide” refers to the structural proteins that form a functional AAV capsid, which can in turn package DNA and infect or transduce a target cell. In some embodiments, a Cap polypeptide comprises a variant AAV capsid protein as disclosed herein. In some embodiments, Cap polypeptides will comprise all of the AAV capsid subunits, but less than all of the capsid subunits may be present as long as a functional capsid is produced. In some embodiments, the nucleic acid sequence encoding Cap polypeptides will be present on a single vector (e.g., plasmid). In some embodiments, the Cap polypeptide comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10 Cap polypeptide, or a variant of any of the foregoing. AAV capsid genes and proteins have been described in, e.g., Knipe DM. et al., (2001) Fields Virology 6(1), which is hereby incorporated by reference in its entirety.

[0185] Cell Density: As used herein, the term “cell density” refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area. For example, cell density may be represented as viable cells (vc) / cm2of culture medium or vc / mL.

[0186] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position or identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in Page 41 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) an appropriate reference polymer. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or labeled) according to the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 100-110, and a second related sequence includes the same motif at positions 110-120, the motif positions of the second related sequence can be said to “correspond to” positions 100-110 of the reference sequence. Those of skill in the art appreciate that corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and / or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, Hhpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE. Two sequences can be identified as corresponding if they are identical or if they share substantial identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) over a length of (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500 or more) units (e.g., nucleotides or amino acids).

[0187] Culture: As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium.

[0188] Fragment: As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, Page 42 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0189] Gene: As used herein, the term “gene” refers to a DNA sequence that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene includes a non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or effect one or more aspects of gene expression (e.g., inducible expression, etc.).

[0190] Gene therapy: As used herein, the term “gene therapy” refers to delivery of a payload with a vector, e.g., a recombinant AAV particle. In some embodiments, gene therapy comprises delivery and / or expression of a payload (e.g., a therapeutic product) to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, gene therapy comprises: insertion, deletion, or editing (e.g., by mutation, by duplication, by demethylation, by methylation, by upregulation, by downregulation, etc.) of specific genomic DNA sequences to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells). Target cells may be from a mammal and / or may be cells in a mammalian subject. Mammals include but are not limited to humans, dogs, cats, cows, sheep, pigs, llamas, etc. In some embodiments, heterologous DNA is transferred to target cells. The heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. In some embodiments, a therapeutic product is a polypeptide encoded by the heterologous DNA. In some embodiments, a therapeutic product is an RNA encoded by the heterologous DNA. In some embodiments, a therapeutic product is a polypeptide and an RNA encoded by the heterologous DNA. Additionally or alternatively, the heterologous DNA may in some manner mediate expression of DNA that Page 43 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) encodes the therapeutic product, or it may encode a product, such as a polypeptide or RNA that in some manner mediates or modulates, directly or indirectly, expression of a therapeutic product. Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression. Such an inhibitor or repressor or other modulator can be a polypeptide, peptide, or nucleic acid (e.g., DNA or RNA). Gene therapy may include in vivo or ex vivo techniques. In some embodiments, viral and non-viral based gene transfer methods can be used to introduce a nucleic acid encoding a polypeptide of interest or to introduce a therapeutic nucleic acid into mammalian cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as poloxamers or liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson WF., (1992) Science 256(5058): pp.808- 813; Miller AD., (1992) Nature 357(6378): pp.455-460; Feuerbach FJ. Et al., (1996) Kidney Int. 49(6): pp.1791-1794; Urnov FD. Et al., (2010) Nat. Rev Genet.11(9): pp.636-646; and Collins M. et al., (2015) Proc Biol Sci.282(1821), each of which is hereby incorporated by reference in its entirety.

[0191] Host Cell: As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence).

[0192] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or Page 44 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0193] Improve, increase, inhibit, or reduce: As used herein the terms “improve”, “increase,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample, e.g., of a culture medium) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may Page 45 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0194] Medium: As used herein, the terms “medium,” “culture medium,” and “growth medium” refer to a solution comprising nutrients to nourish cells (e.g., growing cells, e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for survival and / or minimal growth. The solution can also comprise components that enhance survival and / or growth above the minimal rate, including hormones and growth factors. The solution can be formulated to a pI and concentration of one or more salts that are optimal for cellular survival and / or proliferation. For example, the medium can also be a “defined medium” or “chemically defined medium,” e.g., a serum-free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. One of skill in the art understands a defined medium can comprise recombinant polypeptides, for example, but not limited to, hormones, cytokines, interleukins, and / or other signaling molecules.

[0195] CNS targeting moiety: The phrase “CNS targeting moiety” as used herein refers to a peptide which is effective in targeting a central nervous system (CNS) cell and / or tissue, e.g., an endothelial cell associated with a blood brain barrier, a cell or tissue that is present in the brain, spinal cord, or CNS system. In some embodiments, a CNS-targeting moiety can target a CNS cell or tissue by: (i) contacting a CNS cell or tissue (e.g., binding to one or more receptors expressed on a CNS cell or tissue); (ii) contacting a cell in contact with a CNS cell or tissue (e.g., binding to one or more receptors expressed on a cell in contact with a CNS cell or tissue); (iii) delivering a payload to a CNS cell or tissue; or (iv) any combination of (i)-(iii). In some embodiments, a CNS cell or tissue comprises: a CNS epithelial cell, an endothelial cell associated with a blood brain barrier, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or a combination thereof. In some embodiments, a CNS tissue comprises a tissue found in a: cortex, thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, deep cerebellar nuclei, or other parts of a brain and / or spinal cord. In some embodiments, a CNS targeting moiety can be conjugated or fused to a payload. In some Page 46 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) embodiments, a CNS targeting moiety can be incorporated into a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a CNS targeting moiety can be inserted in an AAV capsid to form a variant AAV capsid protein as disclosed

[0196] Nucleic acid: The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and single- stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo- deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus-atom bridges (also referred to herein as “internucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. In accordance with the methods and compositions described herein, in some embodiments, an RNA comprises a short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR / Cas guide RNA, microRNA (miRNA), and / or a precursor thereof.

[0197] Payload: As used herein, the term “payload” refers to a nucleic acid sequence of interest (e.g., comprising a sequence that encodes a target payload, such as a target polypeptide or RNA) that is desired to be introduced into a cell, tissue, organ, organism, and / or system comprising cells. A target payload can be a heterologous protein with a therapeutic purpose, e.g., an enzyme or antibody. The target payload can be a heterologous nucleic acid with a Page 47 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR / Cas guide RNA, or a precursor thereof. One of skill in the art will recognize that the target payload can be selected from any heterologous protein or nucleic acid of interest. As used herein, “encode” or “encodes” means directs the expression of or processed into. For example, as used herein, a nucleic acid encodes a polypeptide sequence if it directs the expression of that polypeptide sequence. As another example, as used herein, a nucleic acid precursor (e.g., a pri-miRNA or pre-miRNA) encodes a further processed version of the nucleic acid (e.g., mature miRNA) if it is processed into the further processed version.

[0198] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition comprising rAAV particles that is suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen. In some embodiments, a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to achieve a desired therapeutic effect when administered to a subject or population in need thereof (e.g., by a statistically significant probability). A pharmaceutical composition may be specially formulated for administration in solid or liquid form. In some embodiments, a pharmaceutical composition is formulated for administration by parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection. In some embodiments, a pharmaceutical composition is formulated as a sterile solution or suspension, e.g., in a sustained- release formulation. Pharmaceutical compositions of the disclosure may be formulated for administration by injection or infusion (e.g., subcutaneous, intramuscular, intravenous or epidural injection or infusion). For example, compositions may be formulated for administration by retinal, subretinal, intravitreal, suprachoroidal, intraspinal intracisternal magna, or intrathecal injection or infusion. In some embodiments, a pharmaceutical composition is intended and suitable for administration to a human subject. In some embodiments, a pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free). Formulations of the pharmaceutical compositions may include, but are not limited to, formulations for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., targeted for buccal, sublingual, and systemic absorption), boluses, Page 48 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) powders, granules, pastes for application to the tongue; topical application, such as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0199] Polypeptide: The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0200] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, Page 49 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0201] Recombinant AAV (rAAV) particle: A “recombinant AAV particle”, or “rAAV particle,” as used herein, refers to a transduction-competent, replication-defective viral particle comprising an AAV protein shell encapsulating a payload that is flanked on both sides by ITRs. An AAV particle is produced in a suitable host cell (e.g., a HEK293 cell). For example, the host cell is transfected with at least one vector encoding one or more helper polypeptides and nucleic acids (e.g., Ad2 helper polypeptides and nucleic acids), at least one Rep polypeptide, at least one Cap polypeptide, and at least one payload (e.g., for polypeptide expression or a therapeutic nucleic acid), such that the host cell is capable of producing the Rep and Cap polypeptides necessary for packing the rAAV particle. rAAV particles may be used for subsequent gene delivery.

[0202] Rep polypeptide: The term “Rep polypeptide”, as used herein, refers to the AAV non-structural proteins that mediate AAV replication for the production of AAV particles. The AAV replication genes and proteins have been described in, e.g., Knipe 2001, which is hereby incorporated by reference in its entirety.

[0203] Seeding: The term “seeding” as used herein refers to the process of providing a cell culture to a vessel (e.g., a bioreactor or culture flask). For example, the process of providing a cell culture may include propagation of the cells in another bioreactor or vessel before providing to the bioreactor or other vessel. The cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term “seeding” refers to providing any number of cells, including a single cell.

[0204] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory Page 50 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a neurological disease or disorder or a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0205] Titer: As used herein, the term “titer” refers to the quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume.

[0206] Transfection: As used herein, the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells (e.g., mammalian cells). For example, transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and / or transfection based on cationic polymers (e.g., DEAE-dextran or polyethylenimine). In some embodiments, viral-based transfection is also referred to herein as transduction.

[0207] Treating: As used herein, the term “treating” refers to providing treatment, e.g., providing any type of medical or surgical management of a subject. The treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition. “Prevent” refers to causing a disease, disorder, condition, or Page 51 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering an agent to the subject following the development of one or more symptoms or manifestations indicative of a condition, disease, or disorder, e.g., in order to reverse, alleviate, reduce the severity of, and / or inhibit or prevent the progression of the condition and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the condition. A composition comprising rAAV particles of the disclosure can be administered to a subject who has developed a disorder or is at increased risk of developing such a disorder relative to a member of the general population. A composition of the disclosure can be administered prophylactically or before development of any symptom or manifestation of the condition. Typically, in this case, the subject will be at risk of developing the condition.

[0208] Variant: As used herein in the context of molecules, e.g., nucleic acids or polypeptides, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element that comprises a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element that comprises a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some Page 52 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0209] Vector: As used herein, the term “vector” refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into Page 53 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) a cell. By way of non-limiting example, one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be packaged into a viral capsid and can be transferred into another cell and / or organism. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0210] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacture’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook J. et al., (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press 2ndedition, which is incorporated herein by reference in its entirety.

[0211] VP: As used herein, the term “VP” refers to an AAV VP1 capsid protein, an AAV VP2 capsid protein, an AAV VP3 capsid protein, or variants or fragments or combinations of any of the foregoing. The term “capsid protein” is used interchangeably herein with VP. The numbering used herein in describing exemplary locations of peptide insertions in VP1, VP2 or VP3 are used relative to AAV VP1 numbering. For example VP1, VP2 and VP3 of the AAV9 capsid protein correspond to amino acids 1 to 736 of VP1, amino acids 138 to 736 of VP1 and amino acids 203 to 736 of VP1, respectively. Thus, reference to a peptide insertion between positions 588 and 589 in an AAV capsid variant refers to positions 588 and 589 in VP1, VP2 or VP3 relative to VP1 numbering. Those with knowledge in the pertinent field would be able to readily ascertain the corresponding position in VP2 and VP3, e.g., by comparing the sequences of VP1, VP2 and VP3 of the parental AAV capsid proteins using methods known in the field such as sequence alignment. In some embodiments, a VP capsid protein is a VP1 capsid protein. Page 54 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) In some embodiments, a VP capsid protein is a VP2 capsid protein. In some embodiments, a VP capsid protein is a VP3 capsid protein. In some embodiments, a VP protein comprises a peptide insertion disclosed herein.

[0212] Variant AAV capsid protein: As used herein, the term “variant AAV capsid protein” refers to a VP capsid protein (e.g., a VP1, VP2, or VP3) comprising a peptide insertion relative to a corresponding parental AAV capsid protein (e.g., a parental VP1, VP2, or VP3). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0213] The present disclosure provides, inter alia, improved recombinant adeno- associated virus (rAAV) particles that can be used for targeting cells or tissue, e.g., CNS cells and / or tissue. Safe and efficient therapeutic payload delivery to a CNS cell and / or tissue remains a major challenge in gene therapy. Recombinant adeno-associated viruses (rAAVs) have emerged as some of the most promising vectors for in vivo gene therapy, and are currently under clinical evaluation for a number of disorders including CNS disorders. However, naturally occurring AAV capsids sub-optimally target CNS cells or tissue, and require extremely high doses to achieve minimum effective transgene expression. This poses daunting manufacturing challenges as well as safety concerns.

[0214] The present disclosure is based, in part, on the discovery that AAV tropism to certain cells and / or tissue can be obtained by inserting a short peptide into an AAV capsid to generate a variant AAV capsid protein and direct said variant AAV capsid protein to certain cells and / or tissue. Without wishing to be bound by any particular theory, in some embodiments, AAV tropism to certain cells and / or tissue can be obtained with a variant AAV capsid protein which binds to one or more cell surface receptors such as hTfR1 or a variant or a fragment thereof.

[0215] Further without wishing to be bound by theory, binding of a variant AAV capsid protein to hTfR1 or a variant or a fragment thereof, delivers a variant AAV capsid protein to said cell and allows for a variant AAV capsid protein to transcytose said cell (e.g., to be transported from one surface of said cell to another surface of said cell). In some embodiments, transcytosis of a variant AAV capsid protein across a cell by binding to a hTfR1 delivers a variant AAV capsid protein to an organ, to an intersitial space, and / or to an interstitial fluid allowing for Page 55 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) transduction of a cell and / or tissue. In some embodiments, delivery of a variant AAV capsid protein to an organ, to an intersitial space, and / or to an interstitial fluid results in transduction of a cell or tissue.

[0216] For example, when a variant AAV capsid protein is directed to an endothelial cell which expresses a hTfR1 or a variant or a fragment thereof, said variant AAV capsid protein can bind to and transcytose the endothelial cell and / or transduce the endothelial cell. In some embodiments, a variant AAV capsid protein that transcytoses an endothelial cell can be delivered to an organ, an intersitial space, and / or an interstitial fluid.

[0217] As another example example, when a variant AAV capsid protein is directed to an endothelial cell which is part of, or forms a blood brain barrier (e.g., by binding to a hTfR1 or a fragment or variant thereof), said variant AAV capsid protein can bind to and transcytose the endothelial cell and be delivered to the brain. In some embodiments, a variant AAV capsid protein delivered to a brain transduces one or more cells in a brain. In some embodiments, a variant AAV capsid protein directed to an endothelial cell transducse an endothelial cell.

[0218] Additionally, this disclosure provides the discovery that AAV CNS cell and / or tissue tropism can be obtained by inserting a short peptide into an AAV capsid to direct said AAV capsid to a CNS cell and / or tissue. In some embodiments, a variant AAV capsid protein can be directed to a CNS cell and / or tissue (e.g., an endothelial cell associated with a blood brain barrier) by binding to one or more receptors expressed on endothelial cells). This disclosure is also the first to disclose that such variant AAV capsid proteins can be identified by selecting for binding and / or recognition to receptors that are expressed by CNS cell and / or tissue (e.g., an endothelial cell associated with a blood brain barrier). For example, the human transferrin receptor (hTfR1), is a receptor that has been shown to have a role in receptor- mediated transcytosis (RMT) across the blood brain barrier (see, e.g., Bien-Ly, Nga et al. The Journal of experimental medicine vol.211, 2 (2014): pages 233-44; and InternationalApplication PCT / US2019 / 068078 filed on December 20, 2019 the entire contents of each of which is hereby expressly incorporated by reference).

[0219] Accordingly, disclosed herein are technologies for identifying variant AAV capsid proteins with CNS cell and / or tissue tropism by, e.g., binding to hTfR1 or variants or fragments thereof. Also disclosed herein are novel variant AAV capsid proteins that have Page 56 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) enhanced CNS cell and / or tissue tropism for example by binding to hTfR1 or variants or fragments thereof.

[0220] In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein bind to and / or recognize a target on a CNS cell and / or tissue. Without wishing to be bound by any particular theory, in some embodiments, rAAV particles comprising a variant capsid comprising a peptide insertion disclosed herein can enhance vector attachment, internalization, transcytosis, and / or payload expression in a CNS cell and / or tissue. Adeno-associated viruses (AAVs)

[0221] Adeno-associated viruses (AAVs) are small, nonenveloped, single-stranded DNA (ssDNA) viruses that belong to the Parvoviridae family. At least twelve distinct AAV serotypes have been identified from human and nonhuman primate sources (see DiMattia M.A. et al., (2012) J. Virology 86(12): pp.6947-6958, the entire contents of which is hereby incorporated by reference)(hereinafter “DiMattia 2012”). AAV9 is one of the human AAV serotypes that has enhanced transduction efficiency in cardiac and skeletal muscle, liver tissue, pancreatic tissue, and the eye compared to other serotypes (DiMattia 2012).

[0222] The AAV wild-type genome contains at least three genes, rep, cap and X (Büning H. et al., (2019) Molecular Therapy: Methods & Clinical Development vol.12 pages 248-265). The cap gene encodes for viral proteins VP1, VP2, and VP3, and assembly-activating protein (AAP). All three VPs (i.e., viral proteins) are capsid monomers.

[0223] Transcription of the cap gene results in two messenger RNA: a messenger RNA which encodes VP1 and a messenger RNA which encodes VP2 and VP3 (as described in Warrington KH et al., (2004) Journal of Virology volume 78(12) pages 6595-6609). VP1, VP2, and VP3 are present at ratios of 1:1:10, respectively. The VP3 region is observed in all capsid structures of AAV serotypes that have been studied (DiMattia 2012).

[0224] VP proteins comprise beta strands, alpha helical regions, and structurally variable regions (VRs) in the surface loops which connect the beta strands. Without wishing to be bound by any particular theory, it is believed that differences in sequence and / or conformations of VRs contribute to the variability in cellular tropism, differences in tissue transduction efficiency, and / or antigenic reactivity among different AAV serotypes. In some embodiments, differences in Page 57 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) VR sequence and / or structure among different AAV serotypes allow for differential recognition of cell surface glycans and / or tissue specific protein or lipid receptor interaction for internalization.

[0225] Wild type AAV9 (WT AAV9) has nine variable regions VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX (DiMattia 2012, see also Table 3 therein). AAV9 VR-I encompasses amino acid positions 262-269. AAV VR-II encompasses amino acid positions 327-332 and has a role, e.g., in genome packaging. AAV9 VR-III encompasses amino acid positions 382-386. AAV9 VR-IV encompasses amino acid positions 452-460 and has a role, e.g., in liver transduction and / or a delayed blood clearance phenotype. AAV9 VR-V encompasses amino acid positions 488-505 and has a role, e.g., in LamR receptor binding, liver and / or muscle-specific transduction, and / or a delayed blood clearance phenotype. AAV9 VR-VI encompasses amino acid positions 527-539 and has a role, e.g., in LamR receptor binding, and / or a delayed blood clearance phenotype. AAV9 VR-VII encompasses amino acid positions 545-558 and has a role, e.g., in liver transduction and / or delayed blood clearance phenotype. AAV9 VR- VIII encompasses amino acid positions 581-593 and has a role, e.g., in LamR receptor binding and / or transduction. AAV9 VR-IX encompasses amino acid positions 704-714 and has a role, e.g., in heart tropism, melanoma tropism and / or altered tropism.

[0226] In some embodiments, a rAAV particle disclosed herein is a recombinant AAV (rAAV) particle. In some embodiments, a rAAV particle comprises a variant AAV9 capsid protein comprising a peptide insertion disclosed herein. In some embodiments, a peptide insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of a parental AAV capsid protein.

[0227] In some embodiments, a parental AAV capsid protein comprises the sequence of a wildtype AAV capsid protein, or a sequence having at least 95% identity to the sequence of a wildtype AAV capsid protein, or a sequence having no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having no more than 1, no more than 2, no Page 58 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein.

[0228] In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to the sequence of a wildtype AAV capsid protein and one or more mutations, e.g., as disclosed herein.

[0229] In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0230] In some embodiments, a parental AAV capsid protein is other than an AAV9 capsid protein and comprises one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) at a position of VP1, VP2 or VP3 corresponding to position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein.

[0231] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.

[0232] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any Page 59 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0233] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (b) 446 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (c) 470 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3 of an AAV9 capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein; (f) 591 and 621 of a VP1, VP2 or VP3 of an AAV9 capsid protein; or (g) any combination or all of (a)-(f).

[0234] In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVhu68 capsid protein, or an AAVrh10 capsid protein.

[0235] In some embodiments, a parental AAV capsid protein comprises: an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises: the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. Page 60 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0236] In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.

[0237] In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. Exemplary mutations including liver de-targeting mutations are disclosed in Pulicherla N. et al., (2011) Molecular Therapy volume 19, pages 1070-1078, the entire contents of which are hereby incorporated by reference.

[0238] In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de- targeting mutation) comprises a mutation at position 503, e.g., a W503R mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 595, e.g., a W595C mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 457, e.g., a N457H mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 574, e.g., a T574S mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 592, e.g., a Q592L mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 498, e.g., a N498Y or an N498I mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 602, e.g., a L602F mutation. In some embodiments, a mutation that alters a Page 61 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 468, e.g., a P468T mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 500, e.g., a E500D mutation.

[0239] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.

[0240] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0241] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (b) 446 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (c) 470 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3 of an AAV9 capsid protein; (e) 489 and 545 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (f) 591 and 621 of a VP1, VP2 or VP3 of an AAV9 capsid protein; or (g) any combination or all of (a)-(f).

[0242] In some embodiments, a parental AAV capsid protein comprises: an AAV1 capsid protein. In some embodiments, an AAV1 capsid protein sequence is provided in SEQ ID NO: 2002. Page 62 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0243] In some embodiments, a parental AAV capsid protein comprises: an AAV2 capsid protein. In some embodiments, an AAV2 capsid protein sequence is provided in SEQ ID NO: 2003.

[0244] In some embodiments, a parental AAV capsid protein comprises: an AAV3B capsid protein. In some embodiments, an AAV3B capsid protein sequence is provided in SEQ ID NO: 2010.

[0245] In some embodiments, a parental AAV capsid protein comprises an AAV4 capsid protein. In some embodiments, an AAV4 capsid protein sequence is provided in SEQ ID NO: 2051.

[0246] In some embodiments, a parental AAV capsid protein comprises: an AAV5 capsid protein. In some embodiments, an AAV5 capsid protein sequence is provided in SEQ ID NO: 2004.

[0247] In some embodiments, a parental AAV capsid protein comprises: an AAV6 capsid protein. In some embodiments, an AAV6 capsid protein sequence is provided in SEQ ID NO: 2005.

[0248] In some embodiments, a parental AAV capsid protein comprises an AAV7 capsid protein. In some embodiments, an AAV7 capsid protein sequence is provided in SEQ ID NO: 2052.

[0249] In some embodiments, a parental AAV capsid protein comprises: an AAV8 capsid protein. In some embodiments, an AAV8 capsid protein sequence is provided in SEQ ID NO: 2006.

[0250] In some embodiments, a parental AAV capsid protein comprises an AAV10 capsid protein. In some embodiments, an AAV10 capsid protein sequence is provided in SEQ ID NO: 2053.

[0251] In some embodiments, a parental AAV capsid protein comprises an AAV11 capsid protein. In some embodiments, an AAV11 capsid protein sequence is provided in SEQ ID NO: 2054. Page 63 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0252] In some embodiments, a parental AAV capsid protein comprises an AAV12 capsid protein. In some embodiments, an AAV12 capsid protein sequence is provided in SEQ ID NO: 2055.

[0253] In some embodiments, a parental AAV capsid protein comprises an AAV13 capsid protein. In some embodiments, an AAV13 capsid protein sequence is provided in SEQ ID NO: 2056.

[0254] In some embodiments, a parental AAV capsid protein comprises an AAVhu68 capsid protein. In some embodiments, an AAVhu68 capsid protein sequence is provided in SEQ ID NO: 2057.

[0255] In some embodiments, a parental AAV capsid protein comprises an AAVrh10 capsid protein. In some embodiments, an AAVrh10 capsid protein sequence is provided in SEQ ID NO: 2058.

[0256] In some embodiments, a peptide insertion is in VR-I of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0257] In some embodiments, a peptide insertion is in VR-II of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0258] In some embodiments, a peptide insertion is in VR-III of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0259] In some embodiments, a peptide insertion is in VR-IV of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0260] In some embodiments, a peptide insertion is in VR-V of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10. Page 64 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0261] In some embodiments, a peptide insertion is in VR-VI of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0262] In some embodiments, a peptide insertion is in VR-VII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0263] In some embodiments, a peptide insertion is in VR-VIII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0264] In some embodiments, a peptide insertion is in VR-IX of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0265] In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV5, AAV6 or AAV8 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding positions in the capsid proteins of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10capsid protein.

[0266] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0267] In some embodiments, a peptide insertion is in a VP (e.g., VP1, VP2, and / or VP3) of a parental AAV capsid protein.

[0268] In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of a parental AAV capsid protein (e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10).

[0269] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein. Page 65 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0270] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein.

[0271] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein.

[0272] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein.

[0273] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0274] In some embodiments, a peptide insertion site is located between two adjacent amino acids in VR-VIII of a parental AAV capsid protein.

[0275] In some embodiments, a peptide insertion site is located between two non- adjacent amino acids in VR-VIII of a parental AAV capsid protein.

[0276] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein.

[0277] In some embodiments, insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein.

[0278] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid protein, e.g., as compared to a WT AAV9 capsid protein. In some embodiments, an AAV9 WT capsid protein sequence is provided in SEQ ID NO: 2001. In some embodiments, a variant AAV9 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV9 capsid protein.

[0279] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV1 capsid protein, e.g., as compared to a WT AAV1 capsid protein. In some embodiments, an AAV1 WT capsid protein is provided in SEQ ID NO: 2002. In some embodiments, a variant AAV1 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV1 capsid. Page 66 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0280] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV2 capsid protein, e.g., as compared to a WT AAV2 capsid protein. In some embodiments, an AAV2 WT capsid protein is provided in SEQ ID NO: 2003. In some embodiments, a variant AAV2 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV2 capsid protein.

[0281] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV3B capsid protein, e.g., as compared to a WT AAV3B capsid protein. In some embodiments, an AAV3B WT capsid protein is provided in SEQ ID NO: 2010. In some embodiments, a variant AAV3B capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV3B capsid protein.

[0282] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV5 capsid protein, e.g., as compared to a WT AAV5 capsid protein. In some embodiments, an AAV5 WT capsid protein is provided in SEQ ID NO: 2004. In some embodiments, a variant AAV5 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV5 capsid protein.

[0283] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV6 capsid protein, e.g., as compared to a WT AAV6 capsid protein. In some embodiments, an AAV6 WT capsid protein is provided in SEQ ID NO: 2005. In some embodiments, a variant AAV6 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV6 capsid protein.

[0284] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV8 capsid protein, e.g., as compared to a WT AAV8 capsid protein. In some embodiments, an AAV8 WT capsid protein is provided in SEQ ID NO: 2006. In some embodiments, a variant AAV8 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV8 capsid protein. Page 67 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0285] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV4 capsid protein, e.g., as compared to a WT AAV4 capsid protein. In some embodiments, an AAV4 WT capsid protein is provided in SEQ ID NO: 2051. In some embodiments, a variant AAV4 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV4 capsid protein.

[0286] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV7 capsid protein, e.g., as compared to a WT AAV7 capsid protein. In some embodiments, an AAV7 WT capsid protein is provided in SEQ ID NO: 2052. In some embodiments, a variant AAV7 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV7 capsid protein.

[0287] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV10 capsid protein, e.g., as compared to a WT AAV10 capsid protein. In some embodiments, an AAV10 WT capsid protein is provided in SEQ ID NO: 2053. In some embodiments, a variant AAV10 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV10 capsid protein.

[0288] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV11 capsid protein, e.g., as compared to a WT AAV11 capsid protein. In some embodiments, an AAV11 WT capsid protein is provided in SEQ ID NO: 2054. In some embodiments, a variant AAV11 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV11 capsid protein.

[0289] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV12 capsid protein, e.g., as compared to a WT AAV12 capsid protein. In some embodiments, an AAV12 WT capsid protein is provided in SEQ ID NO: 2055. In some embodiments, a variant AAV12 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV12 capsid protein. Page 68 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0290] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV13 capsid protein, e.g., as compared to a WT AAV13 capsid protein. In some embodiments, an AAV13 WT capsid protein is provided in SEQ ID NO: 2056. In some embodiments, a variant AAV13 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV13 capsid protein.

[0291] In some embodiments, a rAAV particle disclosed herein comprises a variant AAVhu68 capsid protein, e.g., as compared to a WT AAVhu68 capsid protein. In some embodiments, an AAVhu68 WT capsid protein is provided in SEQ ID NO: 2057. In some embodiments, a variant AAVhu68 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAVhu68 capsid protein.

[0292] In some embodiments, a rAAV particle disclosed herein comprises a variant AAVrh10 capsid protein, e.g., as compared to a WT AAVrh10 capsid protein. In some embodiments, an AAVrh10 WT capsid protein is provided in SEQ ID NO: 2058. In some embodiments, a variant AAVrh10 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAVrh10 capsid protein.

[0293] Additional modifications to an AAV9 capsid protein (not including peptide insertions disclosed herein) are possible including, for example, variants disclosed in International Patent Application WO 2003 / 052052 filed on November 12, 2002, the entire contents of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in WO 2003 / 052052.

[0294] Several other reports disclose modifications to an AAV9 capsid protein, including: Pulicherla N. et al., (2011) Mol Ther.19(6): pp.1070–1078; Wang D. et al., (2018) Mol Ther Methods Clin Dev. (9): pp.234-246; Adachi K. et al., (2014) Nat. Comm. (5): art. 3075; or Bell CL. Et al., (2012) J Virol.86(13): pp.7326–7333, the entire contents each of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein Page 69 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in any of the reports referenced herein. Variant AAV capsid proteins with peptide insertion

[0295] Among other things, disclosed herein, are AAV capsid protein variants, e.g., AAV9 capsid protein variants having one or more peptide insertions, e.g., as disclosed herein.

[0296] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein, e.g., a variant AAV9 capsid protein comprising a peptide insertion disclosed herein.

[0297] In some embodiments, a peptide insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0298] In some embodiments, a peptide insertion is in VR-I of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0299] In some embodiments, a peptide insertion is in VR-II of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0300] In some embodiments, a peptide insertion is in VR-III of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0301] In some embodiments, a peptide insertion is in VR-IV of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0302] In some embodiments, a peptide insertion is in VR-V of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0303] In some embodiments, a peptide insertion is in VR-VI of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0304] In some embodiments, a peptide insertion is in VR-VII of an AAV capsid protein, e.g., an AAV9 capsid protein. Page 70 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0305] In some embodiments, a peptide insertion is in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0306] In some embodiments, VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580-585, amino acids 585-590, amino acids 590-595, or amino acids 595-601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580-581, between amino acids 581-582, between amino acids 582- 583, between amino acids 583-584, between amino acids 584-585, between amino acids 585- 586, between amino acids 586-587, between amino acids 587-588, between amino acids 588- 589, between amino acids 589-590, between amino acids 590-591, between amino acids 591- 592, between amino acids 592-593, between amino acids 593-594, between amino acids 594- 595, between amino acids 595-596, between amino acids 596-597, between amino acids 597- 598, between amino acids 598-599, between amino acids 599-600, or between amino acids 600- 601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0307] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP3 of an AAV9 capsid protein.

[0308] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein. Page 71 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0309] In some embodiments, a peptide insertion site is located between two adjacent amino acids in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0310] In some embodiments, a peptide insertion site is located between two non- adjacent amino acids in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0311] In some embodiments, a peptide insertion is in VR-IX of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0312] In some embodiments, a peptide insertion is in VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0313] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.

[0314] In some embodiments, insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.

[0315] In some embodiments, a recombinant AAV particle (rAAV) disclosed herein comprises a variant AAV capsid protein, e.g., a variant AAV9 capsid protein, comprising a peptide insertion disclosed herein, and one or more sequences of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0316] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising a peptide provided in any one of Tables 1-14. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 1. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 2. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 3. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 4. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 5. In some Page 72 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 6. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 7. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 8. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 9. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 10. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 11. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 12. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 13. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 14.

[0317] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in any one of Tables 1-14; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 1; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 2; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 3; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 4; and (2) one or Page 73 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 5; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 6; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 7; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 8; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 9; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 10; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 11; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 12; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 13; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the peptides provided in Table 14; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein. Page 74 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0318] In some embodiments, a rAAV particle disclosed herein comprising a variant AAV (e.g., AAV9) capsid protein comprising a peptide provided in any one of Tables 1-14 has an at least 1.5 fold enhanced CNS transduction compared to a rAAV particle comprising a corresponding parental AAV (e.g., AAV9) capsid protein, e.g., without a peptide insertion.

[0319] In some embodiments, a rAAV particle disclosed herein comprising a variant AAV (e.g., AAV9) capsid protein comprising a peptide provided in any one of Tables 1-14 can be useful for CNS-targeting, e.g., targeting a rAAV particle comprising a variant AAV (e.g., AAV9) capsid protein to a CNS cell and / or tissue.

[0320] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein does not comprise an additional sequence N-terminal of a peptide sequence provided in any one of Tables 1-14.

[0321] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein does not comprise an additional sequence C-terminal of a peptide sequence provided in any one of Tables 1-14.

[0322] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein does not comprise an additional sequence N-terminal and C-terminal of a peptide sequence provided in any one of Tables 1-14.

[0323] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 1; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0324] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F.

[0325] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1GX2X3X4X5P (SEQ ID NO: 1004), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F. Page 75 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0326] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1TX2X3X4X5P (SEQ ID NO: 1025), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0327] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2EX3X4X5P (SEQ ID NO: 1024), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0328] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3DX4X5P (SEQ ID NO: 1026), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0329] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3EX4X5P (SEQ ID NO: 1022), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0330] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1GX2X3X4X5P (SEQ ID NO: 1004), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0331] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1TX2X3X4X5P (SEQ ID NO: 1025), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0332] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3EX4X5P (SEQ ID NO: 1022), wherein Page 76 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0333] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2EX3X4X5P (SEQ ID NO: 1024), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0334] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3DX4X5P (SEQ ID NO: 1026), wherein X1is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

[0335] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is R, X2is chosen from A, G, N, S, V or T, X3is chosen from A, Y, D, P, H or E, X4is chosen from D, S, Y, H, or E, X5is chosen from V, A, H, or S, and X6is Y.

[0336] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 1. Table 1: Exemplary peptide insertions encompassed by SEQ ID NO: 1 SEQ ID NO Peptide 42RAADVYPPage 77 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0337] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 42.

[0338] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 42.

[0339] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 43.

[0340] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 43.

[0341] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 46.

[0342] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 46.

[0343] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 47.

[0344] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 47.

[0345] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 49.

[0346] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 49.

[0347] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 51.

[0348] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 51.

[0349] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 53. Page 78 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0350] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 53.

[0351] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 54.

[0352] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 54.

[0353] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 55.

[0354] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 55.

[0355] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 56.

[0356] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 56.

[0357] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 58.

[0358] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 58.

[0359] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 59.

[0360] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 59.

[0361] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 60.

[0362] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 60. Page 79 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0363] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 62.

[0364] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 62.

[0365] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 466.

[0366] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 466.

[0367] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 496.

[0368] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 496.

[0369] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RFKTGYP (SEQ ID NO: 966).

[0370] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RFKTGYP (SEQ ID NO: 966).

[0371] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RPNREYP (SEQ ID NO: 968).

[0372] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RPNREYP (SEQ ID NO: 968). Page 80 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0373] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RDVHKYP (SEQ ID NO: 969).

[0374] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RDVHKYP (SEQ ID NO: 969).

[0375] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RESKIYP (SEQ ID NO: 970).

[0376] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RESKIYP (SEQ ID NO: 970).

[0377] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RQPVQYP (SEQ ID NO: 971).

[0378] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RQPVQYP (SEQ ID NO: 971).

[0379] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RRKEEYP (SEQ ID NO: 972). Page 81 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0380] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RRKEEYP (SEQ ID NO: 972).

[0381] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RERNMYP (SEQ ID NO: 973).

[0382] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RERNMYP (SEQ ID NO: 973).

[0383] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RPNGEYP (SEQ ID NO: 974).

[0384] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RPNGEYP (SEQ ID NO: 974).

[0385] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTQRDYP (SEQ ID NO: 975).

[0386] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTQRDYP (SEQ ID NO: 975). Page 82 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0387] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RAEIQYP (SEQ ID NO: 976).

[0388] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RAEIQYP (SEQ ID NO: 976).

[0389] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise READRYP (SEQ ID NO: 977).

[0390] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not READRYP (SEQ ID NO: 977).

[0391] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RPHKEYP (SEQ ID NO: 978).

[0392] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RPHKEYP (SEQ ID NO: 978).

[0393] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTLDRYP (SEQ ID NO: 979). Page 83 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0394] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTLDRYP (SEQ ID NO: 979).

[0395] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RDGSSYP (SEQ ID NO: 980).

[0396] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RDGSSYP (SEQ ID NO: 980).

[0397] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RSNEPYP (SEQ ID NO: 981).

[0398] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RSNEPYP (SEQ ID NO: 981).

[0399] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RNNSDYP (SEQ ID NO: 982).

[0400] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RNNSDYP (SEQ ID NO: 982). Page 84 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0401] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RDGLRYP (SEQ ID NO: 983).

[0402] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RDGLRYP (SEQ ID NO: 983).

[0403] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RLAVNYP (SEQ ID NO: 984).

[0404] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RLAVNYP (SEQ ID NO: 984).

[0405] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RIAVGYP (SEQ ID NO: 985).

[0406] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RIAVGYP (SEQ ID NO: 985).

[0407] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not ILRCVCDYP (SEQ ID NO: 986). Page 85 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0408] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not DVRSGNFYP (SEQ ID NO: 987).

[0409] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not CRVTITYPT (SEQ ID NO: 988).

[0410] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not MVRFAPAYP (SEQ ID NO: 989).

[0411] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not PREFEWYPH (SEQ ID NO: 990).

[0412] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not DRLRDDYPC (SEQ ID NO: 991).

[0413] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RNPRDYPMF (SEQ ID NO: 992).

[0414] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not LIRRHCHYP (SEQ ID NO: 993). Page 86 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0415] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RNQRTYPAL (SEQ ID NO: 994).

[0416] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTNEQYPQH (SEQ ID NO: 995).

[0417] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RLGPGYP (SEQ ID NO: 996).

[0418] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RLGPGYP (SEQ ID NO: 996).

[0419] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTKEVYP (SEQ ID NO: 1014).

[0420] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTKEVYP (SEQ ID NO: 1014).

[0421] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTNEVYP (SEQ ID NO: 1015). Page 87 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0422] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTNEVYP (SEQ ID NO: 1015).

[0423] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTREVYP (SEQ ID NO: 1016).

[0424] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTREVYP (SEQ ID NO: 1016).

[0425] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTAEVYP (SEQ ID NO: 1017).

[0426] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTAEVYP (SEQ ID NO: 1017).

[0427] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RMEEVYP (SEQ ID NO: 1018).

[0428] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RMEEVYP (SEQ ID NO: 1018). Page 88 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0429] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise REEEVYP (SEQ ID NO: 1019).

[0430] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not REEEVYP (SEQ ID NO: 1019).

[0431] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RDEEVYP (SEQ ID NO: 1020).

[0432] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RDEEVYP (SEQ ID NO: 1020).

[0433] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTYEVYP (SEQ ID NO: 1021).

[0434] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTYEVYP (SEQ ID NO: 1021).

[0435] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RNEDVYP (SEQ ID NO: 527). Page 89 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0436] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RNEDVYP (SEQ ID NO: 527).

[0437] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RREDVYP (SEQ ID NO: 553).

[0438] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RREDVYP (SEQ ID NO: 553).

[0439] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise REEDVYP (SEQ ID NO: 793).

[0440] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not REEDVYP (SEQ ID NO: 793).

[0441] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RHEDVYP (SEQ ID NO: 873).

[0442] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RHEDVYP (SEQ ID NO: 873). Page 90 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0443] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RGEDVYP (SEQ ID NO: 495).

[0444] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RGEDVYP (SEQ ID NO: 495).

[0445] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTDDVYP (SEQ ID NO: 531).

[0446] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTDDVYP (SEQ ID NO: 531).

[0447] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTHDVYP (SEQ ID NO: 518).

[0448] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTHDVYP (SEQ ID NO: 518).

[0449] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTNDVYP (SEQ ID NO: 601). Page 91 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0450] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTNDVYP (SEQ ID NO: 601).

[0451] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTSDVYP (SEQ ID NO: 781).

[0452] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTSDVYP (SEQ ID NO: 781).

[0453] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion does not comprise RTYDVYP (SEQ ID NO: 619).

[0454] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F, with the proviso that the peptide insertion is not RTYDVYP (SEQ ID NO: 619).

[0455] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F with the proviso that the peptide insertion is not RFKTGYP (SEQ ID NO: 966), RPNREYP (SEQ ID NO: 968), RDVHKYP (SEQ ID NO: 969), RESKIYP (SEQ ID NO: 970), RQPVQYP (SEQ ID NO: 971), RRKEEYP (SEQ ID NO: 972), RERNMYP (SEQ ID NO: 973), RPNGEYP (SEQ ID NO: 974), RTQRDYP (SEQ ID NO: 975), RAEIQYP (SEQ ID NO: 976), READRYP (SEQ ID NO: 977), RPHKEYP (SEQ ID NO: 978), RTLDRYP (SEQ ID NO: 979), RDGSSYP (SEQ ID NO: 980), RSNEPYP (SEQ ID NO: 981), RNNSDYP (SEQ ID NO: 982), RDGLRYP (SEQ ID NO: 983), RLAVNYP (SEQ ID NO: 984), or RIAVGYP (SEQ ID NO: 985),Attorney Docket No.: 2011256-2513 (P1829PCT01) ILRCVCDYP (SEQ ID NO: 986), DVRSGNFYP (SEQ ID NO: 987), CRVTITYPT (SEQ ID NO: 988), MVRFAPAYP (SEQ ID NO: 989), PREFEWYPH (SEQ ID NO: 990), DRLRDDYPC (SEQ ID NO: 991), RNPRDYPMF (SEQ ID NO: 992), LIRRHCHYP (SEQ ID NO: 993), RNQRTYPAL (SEQ ID NO: 994), RTNEQYPQH (SEQ ID NO: 995), RLGPGYP (SEQ ID NO: 996), RTKEVYP (SEQ ID NO: 1014), RTNEVYP (SEQ ID NO: 1015), RTREVYP (SEQ ID NO: 1016), RTAEVYP (SEQ ID NO: 1017), RMEEVYP (SEQ ID NO: 1018), REEEVYP (SEQ ID NO: 1019), RDEEVYP (SEQ ID NO: 1020), RTYEVYP (SEQ ID NO: 1021), RNEDVYP (SEQ ID NO: 527), RREDVYP (SEQ ID NO: 553), REEDVYP (SEQ ID NO: 793), RHEDVYP (SEQ ID NO: 873), RGEDVYP (SEQ ID NO: 495), RTDDVYP (SEQ ID NO: 531), RTHDVYP (SEQ ID NO: 518), RTNDVYP (SEQ ID NO: 601), RTSDVYP (SEQ ID NO: 781), or RTYDVYP (SEQ ID NO: 619).

[0456] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F with the proviso that the peptide insertion does not comprise RFKTGYP (SEQ ID NO: 966), RPNREYP (SEQ ID NO: 968), RDVHKYP (SEQ ID NO: 969), RESKIYP (SEQ ID NO: 970), RQPVQYP (SEQ ID NO: 971), RRKEEYP (SEQ ID NO: 972), RERNMYP (SEQ ID NO: 973), RPNGEYP (SEQ ID NO: 974), RTQRDYP (SEQ ID NO: 975), RAEIQYP (SEQ ID NO: 976), READRYP (SEQ ID NO: 977), RPHKEYP (SEQ ID NO: 978), RTLDRYP (SEQ ID NO: 979), RDGSSYP (SEQ ID NO: 980), RSNEPYP (SEQ ID NO: 981), RNNSDYP (SEQ ID NO: 982), RDGLRYP (SEQ ID NO: 983), RLAVNYP (SEQ ID NO: 984), or RIAVGYP (SEQ ID NO: 985), or RLGPGYP (SEQ ID NO: 996), RTKEVYP (SEQ ID NO: 1014), RTNEVYP (SEQ ID NO: 1015), RTREVYP (SEQ ID NO: 1016), RTAEVYP (SEQ ID NO: 1017), RMEEVYP (SEQ ID NO: 1018), REEEVYP (SEQ ID NO: 1019), RDEEVYP (SEQ ID NO: 1020), or RTYEVYP (SEQ ID NO: 1021), RNEDVYP (SEQ ID NO: 527), RREDVYP (SEQ ID NO: 553), REEDVYP (SEQ ID NO: 793), RHEDVYP (SEQ ID NO: 873), RGEDVYP (SEQ ID NO: 495), RTDDVYP (SEQ ID NO: 531), RTHDVYP (SEQ ID NO: 518), RTNDVYP (SEQ ID NO: 601), RTSDVYP (SEQ ID NO: 781), or RTYDVYP (SEQ ID NO: 619). Page 93 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0457] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 2; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0458] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1NDYVSY (SEQ ID NO: 3019), wherein X1is any amino acid except Q.

[0459] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a consists of a sequence of X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence a sequence of X1NDYVSY (SEQ ID NO: 3019), wherein X1is any amino acid except Q.

[0460] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1NDYVSY (SEQ ID NO: 3020), wherein X1is chosen from A, R, or V.

[0461] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 2. Table 2: Exemplary peptide insertions encompassed by SEQ ID NO: 2 SEQ ID NO Peptide 7 ANDYVSY

[0462] In some eAAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 7.

[0463] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 7.

[0464] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 50. Page 94 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0465] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 50.

[0466] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 80.

[0467] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 80.

[0468] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1NDYVSY (SEQ ID NO: 2), wherein X1 is any amino acid, with the proviso that the peptide insertion does not comprise QNDYVSY (SEQ ID NO: 617).

[0469] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of X1NDYVSY (SEQ ID NO: 2), wherein X1 is any amino acid, with the proviso that the peptide insertion is not QNDYVSY (SEQ ID NO: 617).

[0470] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 3; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0471] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R.

[0472] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a consists of a sequence of X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R.

[0473] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2NGX3X4G (SEQ ID NO: 3022), wherein X1is chosen from W, or Y, X2is chosen from D or P, X3is chosen from V or N, and X4is chosen from P or R.

[0474] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 3. Table 3: Exemplary peptide insertions encompassed by SEQ ID NO: 3. Page 95 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID NO Peptide 91 WDNGVPG

[0475] In some embo ant AAV capsid protein disclosed herein comprises asequence of SEQ ID NO: 91.

[0476] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 91.

[0477] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 93.

[0478] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 93.

[0479] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 94.

[0480] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 94.

[0481] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 103.

[0482] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 103.

[0483] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 104.

[0484] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 104.

[0485] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 4; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein. Page 96 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0486] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F.

[0487] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F.

[0488] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of RX1DGADV (SEQ ID NO:4), wherein X1is W or Y.

[0489] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 4. Table 4: Exemplary peptide insertions encompassed by SEQ ID NO: 4. SEQ ID NO Peptide 63 RWDGADV

[0490] In some emb nt AAV capsid proteindisclosed herein comprises a sequence of SEQ ID NO: 63.

[0491] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 63.

[0492] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 64.

[0493] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 64.

[0494] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 961; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0495] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein Page 97 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W.

[0496] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W.

[0497] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 1005) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid except C, and X7is F, Y, or W.

[0498] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 1005) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid except C, and X7is F, Y, or W.

[0499] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 3021) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5is any amino acid and X6is any amino acid except S, and X7is F, Y, or W.

[0500] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 3021) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5is any amino acid and X6is any amino acid except S, and X7is F, Y, or W.

[0501] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 3023), wherein X1is chosen from V or Q, X2is chosen from S or N, X3is chosen from E or D, X4is chosen from F, W, or Y, X5is chosen from L, T, M, Q, A, F, E, S, H, V, or I, X6is chosen from Y, T, N, F, M, S, I, A, Q, G, P, H, E, or V, and X7is chosen from F, Y, or W.

[0502] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2X3X4X5X6X7, (SEQ ID NO: 3023), Page 98 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) wherein X1is chosen from V or Q, X2is chosen from S or N, X3is chosen from E or D, X4is chosen from F, W, or Y, X5is chosen from L, T, M, Q, A, F, E, S, H, V, or I, X6is chosen from Y, T, N, F, M, S, I, A, Q, G, P, H, E, or V, and X7is chosen from F, Y, or W.

[0503] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 5. Table 5: Exemplary peptide insertions encompassed by SEQ ID NO: 961. SEQ ID NO Peptide 396 VSEFLTF12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 759 VSEFYTF 856 VSEFLYF

[0504] In some emb nt AAV capsid protein disclosed herein comprises.

[0505] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 396.

[0506] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 473.

[0507] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 473.

[0508] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 474.

[0509] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 474.

[0510] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 475.

[0511] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 475.

[0512] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 476.

[0513] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 476.

[0514] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 477.

[0515] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 477.

[0516] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 479. Page 100 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0517] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 479.

[0518] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 480.

[0519] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 480.

[0520] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 481.

[0521] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 481.

[0522] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 485.

[0523] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 485.

[0524] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 498.

[0525] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 498.

[0526] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 499.

[0527] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 499.

[0528] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 502.

[0529] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 502. Page 101 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0530] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 507.

[0531] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 507.

[0532] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 516.

[0533] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 516.

[0534] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 530.

[0535] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 530.

[0536] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 532.

[0537] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 532.

[0538] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 536.

[0539] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 536.

[0540] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 544.

[0541] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 544.

[0542] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 546. Page 102 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0543] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 546.

[0544] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 548.

[0545] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 548.

[0546] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 554.

[0547] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 554.

[0548] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 563.

[0549] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 563.

[0550] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 568.

[0551] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 568.

[0552] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 574.

[0553] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 574.

[0554] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 576.

[0555] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 576. Page 103 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0556] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 582.

[0557] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 582.

[0558] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 587.

[0559] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 587.

[0560] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 609.

[0561] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 609.

[0562] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 617.

[0563] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 617.

[0564] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 618.

[0565] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 618.

[0566] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 632.

[0567] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 632.

[0568] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 637. Page 104 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0569] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 637.

[0570] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 666.

[0571] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 666.

[0572] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 676.

[0573] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 676.

[0574] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 692.

[0575] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 692.

[0576] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 701.

[0577] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 701.

[0578] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 759.

[0579] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 759.

[0580] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 856.

[0581] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 856. Page 105 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0582] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 962; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0583] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid.

[0584] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid.

[0585] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 3028) wherein X1is any amino acid except C, S, F, or Y.

[0586] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of REDHVX1W, (SEQ ID NO: 3028) wherein X1is any amino acid except C, S, F or Y.

[0587] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 3024), wherein X1is chosen from L, A, S, R, T, M, Q, H, V, G, E, N, I, F, or Y.

[0588] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of REDHVX1W, (SEQ ID NO: 3024), wherein X1is chosen from L, A, S, R, T, M, Q, H, V, G, E, N, I, F, or Y.

[0589] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 6. Table 6: Exemplary peptide insertions encompassed by SEQ ID NO: 962. SEQ ID NO Peptide 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID NO Peptide 503 REDHVQW 508 REDHVHW

[0590] In some emb nt AAV capsid proteindisclosed herein comprises a sequence of SEQ ID NO: 482.

[0591] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 482.

[0592] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 483.

[0593] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 483.

[0594] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 484.

[0595] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 484.

[0596] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 486.

[0597] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 486.

[0598] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 490.

[0599] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 490. Page 107 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0600] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 501.

[0601] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 501.

[0602] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 503.

[0603] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 503.

[0604] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 508.

[0605] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 508.

[0606] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 550.

[0607] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 550.

[0608] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 608.

[0609] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 608.

[0610] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 627.

[0611] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 627.

[0612] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 785. Page 108 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0613] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 785.

[0614] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 833.

[0615] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 833.

[0616] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 894.

[0617] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 894.

[0618] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 920.

[0619] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 920.

[0620] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion does not comprise REDHVSW (SEQ ID NO: 484).

[0621] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion is not REDHVSW (SEQ ID NO: 484).

[0622] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion does not comprise REDHVFW (SEQ ID NO: 894).

[0623] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion is not REDHVFW (SEQ ID NO: 894). Page 109 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0624] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion does not comprise REDHVYW (SEQ ID NO: 920).

[0625] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion is not REDHVYW (SEQ ID NO: 920).In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion does not comprise REDHVSW (SEQ ID NO: 484), REDHVFW (SEQ ID NO: 894), or REDHVYW (SEQ ID NO: 920).

[0626] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid with the proviso that the peptide insertion is not REDHVSW (SEQ ID NO: 484), REDHVFW (SEQ ID NO: 894), or REDHVYW (SEQ ID NO: 920).

[0627] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 963; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0628] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid.

[0629] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid.

[0630] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of SX1ALMHV (SEQ ID NO: 1007) wherein X1is E, Q or P. Page 110 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0631] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of SX1ALMHV (SEQ ID NO: 1007) wherein X1is E, Q or P.

[0632] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2ALMHV (SEQ ID NO: 3027) wherein X1is E, Q or P and X2is any amino acid.

[0633] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2ALMHV (SEQ ID NO: 3027) wherein X1is E, Q or P and X2is any amino acid.

[0634] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of X1X2ALMHV (SEQ ID NO: 3025), wherein X1is chosen from A, S, D, H, R, V, T, I, P, or E, and X2is chosen from Q, E, P, V, W, D, M, F, V, S, I, H, A, Y, or N.

[0635] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of X1X2ALMHV (SEQ ID NO: 3025), wherein X1is chosen from A, S, D, H, R, V, T, I, P, or E, and X2is chosen from Q, E, P, V, W, D, M, F, V, S, I, H, A, Y, or N.

[0636] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 7. Table 7: Exemplary peptide insertions encompassed by SEQ ID NO: 963. SEQ ID NO Peptide 444 AQALMHV12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 727 SHALMHV 737 VQALMHV 739 SAALMHV

[0637] In some emb nt AAV capsid protein disclosed herein comprises asequence o S Q O: .

[0638] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 444.

[0639] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 494.

[0640] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 494.

[0641] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 515.

[0642] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 515.

[0643] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 575.

[0644] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 575.

[0645] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 578.

[0646] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 578.

[0647] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 586. Page 112 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0648] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 586.

[0649] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 600.

[0650] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 600.

[0651] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 623.

[0652] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 623.

[0653] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 631.

[0654] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 631.

[0655] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 705.

[0656] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 705.

[0657] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 711.

[0658] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 711.

[0659] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 716.

[0660] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 716. Page 113 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0661] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 722.

[0662] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 722.

[0663] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 727.

[0664] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 727.

[0665] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 737.

[0666] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 737.

[0667] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 739.

[0668] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 739.

[0669] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 742.

[0670] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 742.

[0671] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 795.

[0672] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 795.

[0673] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 835. Page 114 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0674] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 835.

[0675] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 848.

[0676] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 848.

[0677] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 865.

[0678] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 865.

[0679] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 867.

[0680] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 867.

[0681] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 964; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0682] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid.

[0683] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid.

[0684] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of RGEX1X2MY (SEQ ID NO: 1008) wherein X1and X2are independently any amino acid except C. Page 115 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0685] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of RGEX1X2MY (SEQ ID NO: 1008) wherein X1and X2are independently any amino acid except C.

[0686] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of RGEX1X2MY (SEQ ID NO: 3026), wherein X1is chosen from E, Q, H, T, I, N, Y, M, D, K, A, S, L, V, or R, and X2is chosen from Y, F, M, W, H, L, or E.

[0687] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of RGEX1X2MY (SEQ ID NO: 3026), wherein X1is chosen from E, Q, H, T, I, N, Y, M, D, K, A, S, L, V, or R, and X2is chosen from Y, F, M, W, H, L, or E.

[0688] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 8. Table 8: Exemplary peptide insertions encompassed by SEQ ID NO: 964. SEQ ID NO Peptide 513 RGEEYMY

[0689] In some embnt AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 513. Page 116 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0690] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 513.

[0691] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 538.

[0692] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 538.

[0693] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 573.

[0694] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 573.

[0695] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 590.

[0696] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 590.

[0697] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 595.

[0698] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 595.

[0699] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 605.

[0700] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 605.

[0701] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 624.

[0702] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 624. Page 117 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0703] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 636.

[0704] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 636.

[0705] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 640.

[0706] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 640.

[0707] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 661.

[0708] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 661.

[0709] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 683.

[0710] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 683.

[0711] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 688.

[0712] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 688.

[0713] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 699.

[0714] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 699.

[0715] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 745. Page 118 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0716] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 745.

[0717] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 813.

[0718] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 813.

[0719] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 824.

[0720] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 824.

[0721] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 838.

[0722] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 838.

[0723] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 876.

[0724] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 876.

[0725] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 877.

[0726] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 877.

[0727] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 889.

[0728] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 889. Page 119 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0729] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence of SEQ ID NO: 965; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0730] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of YX1AAGGG (SEQ ID NO: 1001) wherein X1is S or H. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of YHX1AGGG (SEQ ID NO: 1002) wherein X1is R, K, or A.

[0731] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of YX1AAGGG (SEQ ID NO: 1001) wherein X1is S or H. In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein consists of a sequence of YHX1AGGG (SEQ ID NO: 1002) wherein X1is R, K, or A.

[0732] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965), wherein X1is chosen from S or H, and X2is chosen from A, K ,or R.

[0733] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence provided in Table 9. Table 9: Exemplary peptide insertions encompassed by SEQ ID NO: 965. SEQ ID NO Peptide

[0734] In some embnt AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 613. Page 120 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0735] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 613.

[0736] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 649.

[0737] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 649.

[0738] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein comprises a sequence of SEQ ID NO: 717.

[0739] In some embodiments, a peptide insertion in a variant AAV capsid protein disclosed herein consists of a sequence of SEQ ID NO: 717.

[0740] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, with the proviso that the peptide insertion does not comprise YSRAGGG (SEQ ID NO: 107).

[0741] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, with the proviso that the peptide insertion is not YSRAGGG (SEQ ID NO: 107).

[0742] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, with the proviso that the peptide insertion does not comprise YSKAGGG (SEQ ID NO: 649).

[0743] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, with the proviso that the peptide insertion is not YSKAGGG (SEQ ID NO: 649).

[0744] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, with the proviso that the peptide insertion is not YSRAGGG (SEQ ID NO: 107) or YSKAGGG (SEQ ID NO: 649).In some embodiments, a peptide insertion in an AAV capsid Page 121 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) protein disclosed herein comprises a sequence of YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A, with the proviso that the peptide insertion does not comprise YSRAGGG (SEQ ID NO: 107) or YSKAGGG (SEQ ID NO: 649).In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence provided in Table 10; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0745] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence provided in Table 10. Table 10: Exemplary peptide insertions. SEQ ID NO Peptide Fold change 5 AGVAYSV BPage 122 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID NO Peptide Fold change 34 NVDHGNS B12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID NO Peptide Fold change 78 VHAAVVH C12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID NO Peptide Fold change 122 DRNGPRD B12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID NO Peptide Fold change 166 RRYDDSV B

[0746] Fed in AAV9 VR- VIII that had a more than 4-fold enhanced brain enrichment in hTfR1-KI compared to WT mouse brain regardless of Ly6 haplotypes (FDR < 0.2). A designation of “A” indicates peptides with more than 2-fold and up to 16-fold enrichment, “B” indicates peptides with more than 16- fold and up to 64-fold enrichment, and “C” indicates peptides with more than 64-fold enrichment. Page 126 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0747] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence provided in Table 11; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0748] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence provided in Table 11. Table 11: Exemplary peptide insertions SEQ ID Enhancement over Enhancement over NOPeptideAAV9 AAV9

[0749] AAV9 VR-VIII that had a more than 2-fold enhanced CNS- and / or neuronal transduction in hTfR1- KI / Ly6a / amouse brain as compared to WT AAV9, with the specified promoters. As used in Table 11, “enhancement” refers to enhanced transduction with AAV9 having the specified peptides inserted in VR-VIII compared to transduction with WT AAV9. Page 127 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0750] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence provided in Table 12; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0751] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence provided in Table 12. Table 12: Additional exemplary peptide insertions SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI i12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI i12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI i12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI i12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI i12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ Enhancement over hTfR1 dependency ID NOPeptideAAV9 in hTfR1-KI iates peptides inserted in AAV9 VR-VIII that had a more than 2-fold enhanced brain enrichment in hTfR1-KI compared to WT AAV9. The hTfR1 dependency information in the fourth column indicates peptides inserted in AAV9 VR-VIII that had a more than 2-fold enhanced brain enrichment in hTfR1-KI compared to WT mouse brain regardless of Ly6 haplotypes (FDR < 0.1). For all of the fold change information, a designation of “A” indicates peptides with more than 2-fold and up to Page 134 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 16-fold enrichment, “B” indicates peptides with more than 16-fold and up to 64-fold enrichment, and “C” indicates peptides with more than 64-fold enrichment.

[0753] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a sequence provided in Table 13; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0754] In some embodiments, a peptide insertion in a variant AAV (e.g., AAV9) capsid protein disclosed herein comprises a sequence provided in Table 13. Table 13: Exemplary peptide insertions SEQ IDPepthTfR1 dependency hTfR1 dependency NOide(CBh promoter) (hSyn1 promoter)Page 135 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ ID hTfR1 dependency hTfR1 dependency NOPeptide(CBh promoter) (hSyn1 promoter)12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) SEQ IDPeptihTfR1 dependency hTfR1 dependency NOde(CBh promoter) (hSyn1 promoter)

[0755] AAV9 VR-VIII that had a more than 4-fold enhanced CNS- and / or neuronal transduction in hTfR1- KI / Ly6a / amouse brain as compared to WT mouse brain, with the specified promoters.

[0756] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid comprising a peptide insertion disclosed in any one of Tables 1-14 and one or more modifications to an amino acid sequence flanking the peptide insertion site. In some embodiments, one or more modifications are within about 10, e.g., within about 5 amino acids upstream or downstream of the location of the peptide insertion site. Page 146 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01)

[0757] In some embodiments, one or more modifications comprises an insertion, deletion, mutation, or a combination thereof.

[0758] In some embodiments, a variant AAV (e.g., AAV9) capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose.

[0759] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f). Exemplary capsid variants with peptide insertions in VR-VIII

[0760] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises any one of the peptide insertions disclosed herein; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.

[0761] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 1; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV Page 147 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0762] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 2; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV Page 148 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0763] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 3; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV Page 149 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutat...

Claims

Attorney Docket No.: 2011256-2513 (P1829PCT01) CLAIMS We claim:

1. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of: (I) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; (II) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; or (III) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; or (IV) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; or (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; or (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; or (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; or (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

2. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: Page 261 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) (i) the peptide insertion comprises a sequence provided in Table 10, Table 11, Table 12, Table 13, or Table 14; and (ii) the peptide insertion site is in a variable region of the parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

3. The rAAV particle of claim 1 or 2, wherein the insertion site is located between two adjacent amino acids in the variable region of the parental AAV capsid protein.

4. The rAAV particle of any one of the preceding claims, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.

5. The rAAV particle of any one of the preceding claims, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein.

6. The rAAV particle of claim 5, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein.

7. The rAAV particle of any one of the preceding claims, wherein the insertion site is located between amino acids 588 and 589 of VP1 of an AAV9 capsid protein or the corresponding position in VP1, VP2, and / or VP3 of another parental AAV capsid protein.

8. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises a peptide insertion comprising the consensus sequence of any one of SEQ ID NOs: 1-4, or 961-965 and one or more sequences of VP1, VP2, or VP3 of an AAV9 capsid protein. Page 262 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 9. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 1 and one or more sequences of VP1, VP2, or VP3 of an AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 1.

10. The rAAV particle of claim 9, wherein the peptide insertion comprises the sequence of SEQ ID NO: 46 (RGADVYP), SEQ ID NO: 466 (RTEDVYP), or SEQ ID NO: 496 (RTEEVYP).

11. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 2 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 2.

12. The rAAV particle of claim 11, wherein the peptide insertion comprises the sequence of SEQ ID NO: 80 (VNDYVSY).

13. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 3 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 3.

14. The rAAV particle of claim 13, wherein the peptide insertion comprises the sequence of SEQ ID NO: 93 (WPNGVPG), or SEQ ID NO: 91 (WDNGVPG).

15. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 4 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 4. Page 263 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 16. The rAAV particle of claim 15, wherein the peptide insertion comprises the sequence of SEQ ID NO: 63 (RWDGADV).

17. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 961 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 5.

18. The rAAV particle of claim 17, wherein the peptide insertion comprises the sequence of SEQ ID NO: 396 (VSEFLTF).

19. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 962 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 6.

20. The rAAV particle of claim 19, wherein the peptide insertion comprises the sequence of SEQ ID NO: 483 (REDHVAW).

21. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 963 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 7.

22. The rAAV particle of claim 21, wherein the peptide insertion comprises the sequence of SEQ ID NO: 444 (AQALMHV).

23. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 964 and one or more Page 264 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 8.

24. The rAAV particle of any one of claims 1-8, wherein the variant AAV capsid protein comprises a peptide insertion comprising a sequence of SEQ ID NO: 965 and one or more sequences of VP1, VP2, or VP3 of the AAV9 capsid protein, optionally wherein the peptide insertion comprises a sequence provided in Table 9.

25. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein confers at least 1.5-fold increased infectivity and / or transduction of a central nervous system (CNS) cell compared to: the infectivity and / or transduction of the CNS cell by a control AAV particle comprising the corresponding parental AAV capsid protein, or the infectivity and / or transduction of a CNS cell which does not express a receptor recognized by the variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

26. The rAAV particle of claim 25, wherein the CNS cell comprises a CNS epithelial cell, an endothelial cell associated with a blood brain barrier, a nerve cell, a CNS connective tissue cell, a stem cell or progenitor cell, a CNS immune cell, a spinal cord cell, or combinations thereof, optionally wherein: (i) the CNS epithelial cell comprises a cell that lines one or more brain ventricles; (ii) the nerve cell comprises nerve support cells, e.g., astrocytes, glial cells, and Schwann cells;and / or (iii) the CNS connective tissue cell comprises fat cells, meninges cells, or both.

27. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site, optionally wherein the one or more modifications are within about 10 amino acids upstream or downstream of the location of the peptide insertion site, e.g., within about 5 amino acids upstream or downstream of the location of the peptide insertion site. Page 265 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 28. The rAAV particle of any one of the preceding claims wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region, optionally wherein the one or more modifications reduces glycan binding.

29. The rAAV particle of claim 27 or 28, wherein the one or more modifications is at or between amino acids: (a) 271 and 272 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (b) 446 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (c) 470 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (e) 489 and 545 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (f) 591 and 621 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; or (g) any combination of (a)-(f).

30. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein has at least 90% identity relative to a parental AAV capsid protein, optionally wherein percent identity is determined by comparing the sequence of the variant AAV capsid protein without the peptide insertion, with the parental AAV capsid protein.

31. The rAAV particle of any one of the preceding claims, wherein the parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001. Page 266 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 32. The rAAV particle of any one of the preceding claims, wherein the payload is or comprises a polypeptide.

33. The rAAV particle of claim 32, wherein the polypeptide is or comprises: (i) a CRISPR-Cas protein, optionally, wherein the CRISPR-Cas protein is a Type II, Type V or Type VI CRISPR-Cas protein, e.g., a Cas9 protein, a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment of any of the foregoing; (ii) a Zinc finger protein, or a variant or a fragment thereof; (iii) a TALEN protein, or a variant or a fragment thereof; (iv) a base editor, or a variant or a fragment thereof; (v) a prime editor, or a variant or a fragment thereof; (vi) a meganuclease or a variant or a fragment thereof; (vii) an enzyme; (viii) an antibody; and / or (ix) a secreted protein.

34. The rAAV particle of claim 32 or 33, wherein the polypeptide is associated with a CNS disorder.

35. The rAAV particle of any one of claims 1-31, wherein the payload is or comprises an RNA molecule, optionally wherein the RNA molecule is a mRNA, an siRNA, a miRNA, a gRNA, an antisense RNA, a circular RNA, an snRNA, or an aptamer.

36. The rAAV particle of claim 35, wherein the RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a CNS disorder.

37. The rAAV particle of claim 34 or 35, wherein the CNS disorder is chosen from Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome Page 267 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) (De Vivo Syndrome), Tay-Sachs, Spinal Muscular Atrophy, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies, or combinations thereof.

38. The rAAV particle of any one of claims 1-31, wherein the payload is or comprises a DNA molecule.

39. The rAAV particle of any one of the preceding claims, wherein the nucleotide sequence encoding a payload is operably linked to a promoter., optionally wherein the promoter is a CNS promoter.

40. The rAAV particle of claim 39, wherein the CNS promoter is chosen from: a GFAP promoter, a SYN1 promoter, a NSE / RU5’ promoter, a neuroactive peptide cholecystokinin (CCK) promoter, a myelin basic promoter (MBP), a human myelin associated glycoprotein promoter, a phosphate-activated glutaminase (PAG) promoter, a vesicular glutamate transporter (vGLUT) promoter, a glutamic acid decarboxylase (GAD) promoter, Camk2a promoter, TH (tyrosine hydroxylase) promoter, Hb9 promoter, CNP promoter, NES (nestin) promoter, Tub1a promoter, SST (somatostatin) promoter, MeCP2 promoter, or combinations thereof.

41. A pharmaceutical composition comprising: (a) a rAAV particle of any one of the preceding claims; and (b) a pharmaceutically acceptable excipient.

42. A method of delivering a payload to a CNS cell, comprising administering the pharmaceutical composition of claim 41 to the CNS cell.

43. A method of treating a subject having a CNS disorder and / or ameliorating a symptom of a CNS disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 41. Page 268 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 44. The method of claim 43, wherein the CNS disorder is chosen from Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay-Sachs, Spinal Muscular Atrophy, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies, or combinations thereof.

45. The method of any one of claims 42-44, wherein the pharmaceutical composition is administered via a route of administration chosen from: intravenous, intraarterial, intracoronoary, intraparenchymal, subpial, intrathecal, intraocular, intracerebroventricular (ICV), intracisternal magna (ICM), or intramuscular.

46. The method of any one of claims 42-45, wherein the subject is a human.

47. An isolated cell transduced with the rAAV particle of any one of claims 1-40.

48. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) the peptide insertion comprises a sequence of: (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; or (IV) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; or (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; or (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; or Page 269 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; or (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein.

49. An isolated cell comprising the nucleic acid of claim 48.

50. A variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) the peptide insertion comprises a sequence of: (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; or (IV) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; or (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; or (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; or (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; or (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A; and Page 270 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein.

51. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence of (I) X1X2X3X4X5X6P (SEQ ID NO: 1), wherein X1is chosen from R or K, X2, X3, X4and X5are independently any amino acid, and X6is chosen from Y, W, or F; (II) X1NDYVSY (SEQ ID NO: 2), wherein X1is any amino acid; (III) X1X2NGX3X4G (SEQ ID NO: 3), wherein X1is Y, W, or F, X2is any amino acid, X3is V or N, and X4is P or R; or (IV) RX1DGADV (SEQ ID NO:4), wherein X1is Y, W, or F; or (V) X1X2X3X4X5X6X7, (SEQ ID NO: 961) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid, and X7is F, Y, or W; or (VI) REDHVX1W, (SEQ ID NO: 962) wherein X1is any amino acid; or (VII) X1X2ALMHV (SEQ ID NO: 963) wherein X1and X2are independently any amino acid; or (VIII) RGEX1X2MY (SEQ ID NO: 964) wherein X1and X2are independently any amino acid; or (IX) YX1X2AGGG (SEQ ID NO: 965) wherein X1is S or H, and X2is R, K, or A;.

52. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence provided in Table 10, Table 11,Table 12, Table 13, or Table 14.

53. The composition of claim 52, wherein the targeting moiety is conjugated to the payload.

54. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in any one of Table 1, Table 2, Page 271 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, or Table 14. (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

55. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a sequence of at least 4, at least 5, or at least 6 contiguous amino acids of a sequence provided in any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, or Table 14.

56. The rAAV particle of any one of claims 1-9 or 25-40, wherein the peptide insertion is not RFKTGYP (SEQ ID NO: 966), RPNREYP (SEQ ID NO: 968), RDVHKYP (SEQ ID NO: 969), RESKIYP (SEQ ID NO: 970), RQPVQYP (SEQ ID NO: 971), RRKEEYP (SEQ ID NO: 972), RERNMYP (SEQ ID NO: 973), RPNGEYP (SEQ ID NO: 974), RTQRDYP (SEQ ID NO: 975), RAEIQYP (SEQ ID NO: 976), READRYP (SEQ ID NO: 977), RPHKEYP (SEQ ID NO: 978), RTLDRYP (SEQ ID NO: 979), RDGSSYP (SEQ ID NO: 980), RSNEPYP (SEQ ID NO: 981), RNNSDYP (SEQ ID NO: 982), RDGLRYP (SEQ ID NO: 983), RLAVNYP (SEQ ID NO: 984), or RIAVGYP (SEQ ID NO: 985), ILRCVCDYP (SEQ ID NO: 986), DVRSGNFYP (SEQ ID NO: 987), CRVTITYPT (SEQ ID NO: 988), MVRFAPAYP (SEQ ID NO: 989), PREFEWYPH (SEQ ID NO: 990), DRLRDDYPC (SEQ ID NO: 991), RNPRDYPMF (SEQ ID NO: 992), LIRRHCHYP (SEQ ID NO: 993), RNQRTYPAL (SEQ ID NO: 994), RTNEQYPQH (SEQ ID NO: 995), RLGPGYP (SEQ ID NO: 996), RTKEVYP (SEQ ID NO: 1014), RTNEVYP (SEQ ID NO: 1015), RTREVYP (SEQ ID NO: 1016), RTAEVYP (SEQ ID NO: 1017), RMEEVYP (SEQ ID NO: 1018), REEEVYP (SEQ ID NO: 1019), RDEEVYP (SEQ ID NO: 1020), RTYEVYP (SEQ ID NO: 1021), RNEDVYP (SEQ ID NO: 527), RREDVYP (SEQ ID NO: 553), REEDVYP (SEQ ID NO: 793), RHEDVYP (SEQ ID NO: 873), RGEDVYP (SEQ ID NO: 495), RTDDVYP (SEQ ID NO: 531), RTHDVYP (SEQ ID NO: 518), RTNDVYP (SEQ ID NO: 601), RTSDVYP (SEQ ID NO: 781), or RTYDVYP (SEQ ID NO: 619). Page 272 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 57. The rAAV particle of any one of claims 1-8 or 11 or 25-40, wherein the peptide insertion is not QNDYVSY (SEQ ID NO: 617).

58. The rAAV particle of any one of claims 1-8 or 19 or 25-40, wherein the peptide insertion is not REDHVSW (SEQ ID NO: 484), REDHVFW (SEQ ID NO: 894), or REDHVYW (SEQ ID NO: 920).

59. The rAAV particle of any one of claims 1-8 or 24 or 25-40, wherein the peptide insertion is not YSRAGGG (SEQ ID NO: 107) or YSKAGGG (SEQ ID NO: 649).

60. The rAAV particle of any one of claims 1-9 or 25-40, wherein the peptide insertion comprises a sequence of: (i) X1TX2X3X4X5P (SEQ ID NO: 1025), wherein X1 is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F, (ii) X1X2EX3X4X5P (SEQ ID NO: 1024), wherein X1 is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F; (iii) X1X2X3DX4X5P (SEQ ID NO: 1026), wherein X1 is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F; or (iv) X1X2X3EX4X5P (SEQ ID NO: 1022), wherein X1 is chosen from R or K, X2, X3, and X4are independently any amino acid, and X5is chosen from Y, W, or F.

61. The rAAV particle of any one of claims 1-8 or 11 or 25-40, wherein the peptide insertion comprises a sequence of: (i) X1NDYVSY (SEQ ID NO: 3019), wherein X1is any amino acid except Q; or (ii) X1NDYVSY (SEQ ID NO: 3020), wherein X1is chosen from A, R, or V.

62. The rAAV particle of any one of claims 1-8 or 13 or 25-40, wherein the peptide insertion comprises a sequence of: X1X2NGX3X4G (SEQ ID NO: 3022), wherein X1is chosen from W, or Y, X2is chosen from D or P, X3is chosen from V or N, and X4is chosen from P or R. Page 273 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 63. The rAAV particle of any one of claims claims 1-8 or 15 or 25-40, wherein the peptide insertion comprises a sequence of: RX1DGADV (SEQ ID NO: 4), wherein X1is W or Y.

64. The rAAV particle of any one of claims 1-8 or 17 or 25-40, wherein the peptide insertion comprises a sequence of: (i) X1X2X3X4X5X6X7, (SEQ ID NO: 1005) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5and X6are independently any amino acid except C, and X7is F, Y, or W; (ii) X1X2X3X4X5X6X7, (SEQ ID NO: 3021) wherein X1is V or Q, X2is S or N, X3is D or E, X4is F, Y, or W, X5is any amino acid and X6is any amino acid except S, and X7is F, Y, or W; or (iii) X1X2X3X4X5X6X7, (SEQ ID NO: 3023), wherein X1is chosen from V or Q, X2is chosen from S or N, X3is chosen from E or D, X4is chosen from F, W, or Y, X5is chosen from L, T, M, Q, A, F, E, S, H, V, or I, X6is chosen from Y, T, N, F, M, S, I, A, Q, G, P, H, E, or V, and X7is chosen from F, Y, or W.

65. The rAAV particle of any one of claims 1-8 or 19 or 25-40, wherein the peptide insertion comprises a sequence of: (i) REDHVX1W, (SEQ ID NO: 3028) wherein X1is any amino acid except C, S, F or Y; or (ii) REDHVX1W, (SEQ ID NO: 3024), wherein X1is chosen from L, A, S, R, T, M, Q, H, V, G, E, N, I, F, or Y.

66. The rAAV particle of any one of claims 1-8 or 21 or 25-40, wherein the peptide insertion comprises a sequence of: (i) SX1ALMHV (SEQ ID NO: 3007) wherein X1is E, Q or P; (ii) X1X2ALMHV (SEQ ID NO: 3027) wherein X1is E, Q or P and X2is any amino acid; or (iii) X1X2ALMHV (SEQ ID NO: 3025), wherein X1is chosen from A, S, D, H, R, V, T, I, P, or E, and X2is chosen from Q, E, P, V, W, D, M, F, V, S, I, H, A, Y, or N. Page 274 of 276 12609119v1Attorney Docket No.: 2011256-2513 (P1829PCT01) 67. The rAAV particle of any one of claims 1-8 or 23 or 25-40, wherein the peptide insertion comprises a sequence of: (i) RGEX1X2MY (SEQ ID NO: 1008) wherein X1and X2are independently any amino acid except C; or (ii) RGEX1X2MY (SEQ ID NO: 3026), wherein X1is chosen from E, Q, H, T, I, N, Y, M, D, K, A, S, L, V, or R, and X2is chosen from Y, F, M, W, H, L, or E.

68. The rAAV particle of any one of claims 1-8 or 24 or 25-40, wherein the peptide insertion comprises a sequence of: (i) YX1AAGGG (SEQ ID NO: 1001) wherein X1is S or H; (ii) YHX1AGGG (SEQ ID NO: 1002) wherein X1is R, K, or A; or (iii) YX1X2AGGG (SEQ ID NO: 965), wherein X1is chosen from S or H, and X2is chosen from A, K ,or R. Page 275 of 276 12609119v1

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